Case#: N/A. This patient is the only one studied in this paper. Female. Age of Onset: 11 y.o. Age of evaluation: 13 y.o. Origin not specified but looking at the author information, I believe it can be safely inferred that the patient is originally from Japan.

DiseaseAssertion: Autoimmune enteropathy with CTLA4 haploinsufficiency

FamilyInfo: Patient had no family history of autoimmune diseases, except for hypothyroidism in her mother and hyperthyroidism in her sister. The patient’s mother and sister were found to have the same heterozygous mutation, but they showed only thyroid gland dysfunction that was manageable with medication, which suggests incomplete penetrance.

CasePresentingHPOs: HP:0002720 (low IgA), OMIM:188030 (immune thrombocytopenic purpura (ITP)), sometimes accompanied by HP:0001890 (autoimmune hemolytic anemia), HP:0001882 (leukopenia), and ORPHA:56425 (cold agglutinin disease).

CaseHPOFreeText: Persistent watery diarrhea, immunoglobulin A (IgA) deficiency with low IgG2 and IgG4 subclass, recurrent immune thrombocytopenic purpura (ITP).

The patient had CTLA4 haploinsufficiency and was positive for anti-AIE-75 autoantibody but was successfully treated with 6-mercaptopurine (6-MP)

Patient was suspected of having interstitial nephritis

The patient responded well to prednisolone (PSL; 2 mg/kg/day) that was used to treat ITP at every onset, and the treatment was successfully tapered off. However, watery diarrhea appeared during the tapering of PSL while the treatment for the fifth episode of ITP was ongoing.

Her height was 155 cm (third to fifth percentile) and weight was 48 kg (fifth percentile)

In our patient, clinical remission was achieved with 40 mg/day of PSL. Clinical remission was maintained on 6-MP even after cessation of PSL for 4 years.

CaseNotHPOs: N/A.

CaseNotHPOFreeText: After analyzing the patient’s past laboratory data, we found that following cessation of PSL, ITP relapsed in association with the normalization of IgG levels (861–1,747 mg/dL). In addition, we found that serum levels of sIL-2R were extremely elevated at the onset of AIE. There was no manifestation of AIE, ITP, or other autoimmune diseases for 4 years, while the dose of 6-MP was adjusted to keep the levels of IgG and sIL-2R below 700 mg/dL and 1,500 U/mL, respectively (Fig. 3).

Flow cytometry analysis showed reduced frequency of CD4+ CD25+ FOXP3+ Tregs (Fig. 4) [10]. Consistent with clinical remission of AIE, an endoscopy and histological study 3 years after the achievement of remission demonstrated recovery from villous atrophy (Fig. 5). After 4 years of 6-MP administration, we tried to reduce the amount of 6-MP because we were concerning about the risk of secondary malignancies such as malignant lymphoma caused by 6-MP. Unfortunately, it led to the relapse of diarrhea and ITP along with soar up of serum level of rIL-2 receptor (Fig. 3).

CasePreviousTesting: The patient underwent many different types of testing:

None of the viruses, namely, adenovirus, enterovirus, cytomegalovirus, Epstein-Barr virus, norovirus, and rotavirus, were detected in the patient’s stool sample using polymerase chain reaction. In addition, no pathogenic bacteria were cultured from the stool. Besides, a toxin detection test for Clostridium difficile also yielded negative results. A computed tomography scan of her abdomen revealed a small volume of ascites.

An esophagogastroduodenoscopy and a colonoscopy demonstrated prominent atrophy of the villus in the duodenum (Fig. 1A) and terminal ileum. Colon was intact endoscopically (Fig. 1B). Histopathological examinations of the duodenum and ileum showed severe villous atrophy. Crypt apoptosis, and infiltration of lymphoplasmacytes into the lamina propria were seen in the biopsies of duodenum, ileum, and colon (Fig. 1C-​-E).E). Immunostaining of the normal small intestine tissue with the patient’s serum showed positive staining of the brush borders of the intestinal epithelium, indicating the presence of anti-enterocyte autoantibodies in the serum of the patient (Fig. 2A and ​andB).B). Furthermore, autoantibodies against AIE-75 were detected by Western blotting, using a recombinant protein as an antigen (Fig. 2C). The patient was diagnosed with AIE and was administered an increased dose of PSL (40 mg/day) as induction therapy.

Whole-exome sequencing demonstrated a heterozygous missense mutation in the CTLA4 gene. Flow cytometry analysis showed reduced frequency of CD4+ CD25+ FOXP3+ Tregs

GenotypingMethod: Whole-exome sequencing demonstrated a heterozygous missense mutation in the CTLA4 gene

PreviouslyPublished: Yes, PMID: 29729943. Schwab et al.

Variant: NM_001037631.2:c.119T>C

ClinVarID: None found

CAID: CA350138103

gnomAD: None found

SupplementalData: N/A

Note: Not functionally tested using transendocytosis

Case#: N/A. Patient was the only one included in this paper. Female. Age of Onset: 28 y.o. Age of evaluation: 28 y.o. Origin not specified but looking at the author information, I believe it can be safely inferred that the patient is originally from Japan.

DiseaseAssertion: CTLA4 haploinsufficiency in a patient with Epstein–Barr virus-positive diffuse large B-cell lymphoma and subsequent benign lymphadenopathy

FamilyInfo: A missense mutation in exon 2 of the CTLA4 gene (c.251T>C, p.V84A) was found in the patient’s peripheral blood and buccal cell DNA, but not in her parents’ DNA.

Neither of the parents had this mutant allele of CTLA4 (Figure 3A) and the case was considered to be sporadic.

CasePresentingHPOs: HP:0001047 (Atopic dermatitis), HP:0012378 (fatigue), HP:0001945 (fever), HP:0002716 (Lymphadenopathy/swollen lymph nodes), HP:0001744 (splenomegaly).

CaseHPOFreeText: Mild atopic dermatitis, high fever, multiple swollen lymph nodes, systemic lymphadenopathy and multiple bone lesions.

CTLA4 expression decreased in the peripheral regulatory T cells upon stimulation, whereas CTLA4 and PD-1-positive T cell subsets increased, possibly to compensate for the defective CTLA4 function

Although the patient had no history of autoimmune disease or specific infections, her uncommon clinical course led us to perform genetic screening for congenital immune dysfunction, and a missense germline mutation in CTLA4 was identified.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: Immunohistochemistry was performed with antibodies against the following proteins: CD20 (346595, BD Biosciences, San Jose, CA), CD3 (349201, BD Biosciences), CD30 (clone Ber-H2, Roche, Mannheim, Germany), CD15 (Carb-3, Dako, Santa Barbara, CA), Ki-67 (clone MIB-1, Dako), EBV LMP-1 (CS-1-4, Dako), EBV EBNA2 (PE2, Dako), CTLA4 (sc-376016, Santa Cruz Biotechnology, Santa Cruz, CA), and FOXP3 (clone PCH101, eBioscience, San Diego, CA). Immunohistochemistry was performed using an automatic immunostainer (BenchMark, Ventana Medical Systems, Tucson, AZ and BOND-III system, Leica Microsystems, Bannockburn, IL) in accordance with the manufacturer’s instructions.

Epstein–Barr virus detection was performed by in situ hybridization using an EBV-encoded small non-polyadenylated RNA probe on an automated system (Ventana Medical systems for Figure 1B, g, and Leica Microsystems for Figure 2B, e).

Peripheral blood mononuclear cells were separated from the peripheral blood of the patient and two healthy donors using a Ficoll-Paque density gradient (Cedarlane), and total T cells were collected by negative selection using MACS Cell Separation Technology (Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA was extracted using the RNeasy Mini kit, and complementary DNA (cDNA) was synthesized using a SuperScript III First-Star and Synthesis system (Life Technologies, Carlsbad, CA, USA). qRT-PCR was performed using TB Green Premix Ex Taq II (Takara Bio, Otsu, Japan). The primers used for the amplification are listed in Table 1. The expression levels of FOXP3, CTLA4, and PDCD1 were normalized to that of ACTB.

[18F]-Fluorodeoxy-d-glucose positron emission tomography (FDG-PET)-computed tomography (CT) revealed systemic lymphadenopathy, splenomegaly, and multiple bone lesions (Figure 1A). Laboratory data included increases in lactate dehydrogenase (LDH) to 1,127 IU/L (normal range, 117–236 IU/L), soluble interleukin-2 receptor (sIL-2R) to 10,500 U/mL (145–519 U/mL), and β2-microglobulin to 4.6 µg/mL (1.0–1.9 µg/mL). Serum IgG and IgA moderately decreased to 651 mg/dL (870–1,700 mg/dL) and 28 mg/dL (110–410 mg/dL), respectively, whereas IgM was normal (78 mg/dL; normal range 35–220 mg/dL). The patient’s serum was negative for human immunodeficiency virus-1 antibody.

Histological analysis of the biopsied right axillar lymph node first led to a diagnosis of classic Hodgkin lymphoma (cHL), but the diagnosis was later revised to EBV-positive DLBCL with a T-cell-rich large B-cell lymphoma-like pattern (Figure 1B). The large tumor cells were positive for CD20, CD30 (weak, 30%), and EBV-encoded small RNA (EBER)-in situ hybridization (ISH), and negative for CD3 and CD15. Ki-67 was positive in 80% of the tumor cells. The tumor cells expressed EBV latent membrane protein 1 (LMP-1), but lacked EBV nuclear antigen 2 (EBNA2), and exhibited a type 2 latency pattern (Figure 1C). The patient was treated with one cycle of ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) and six cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), and achieved complete remission.

Two years later, the patient developed cervical lymph node swelling (Figure 2A). Although the relapse of DLBCL was suspected, histological analysis of the biopsied cervical lymph node revealed only reactive follicular hyperplasia with a few, small EBER-positive cells (Figure 2B, a-e). The patient had normal blood cell counts with a relatively low lymphocyte count of 590/µL (normal range, 400–3,700/µL) and normal lymphocyte subsets (CD3+ T cells, 72.1%; CD3+CD4+ T cells, 38.5%; CD3+CD8+ T cells, 28.1%; CD56+ NK cells, 12.7%; CD19+ B cells, 15.2%). However, the serum immunoglobulin levels further decreased (IgG 320 mg/dL, IgA 10 mg/dL, IgM 40 mg/dL). The serum EBV-DNA was 190 copies/µg of DNA when evaluated after 1 cycle of ABVD and became negative after the next cycle of chemotherapy. However, it became positive again half a year before the appearance of lymphadenopathy and remained positive at low levels thereafter (20–60 copies/µg of DNA).

Persisting lymphadenopathy with low immunoglobulin levels and serum EBV-DNA positivity led us to consider the possibility of congenital immune dysfunction.

We evaluated CTLA4 expression upon stimulation of peripheral regulatory T cells, which was markedly reduced according to flow cytometry6 (Figure 3B), and the patient was diagnosed with CTLA4 haploinsufficiency.

GenotypingMethod: The patient’s peripheral blood DNA was screened for germline mutations (Kazusa DNA Research Institute, Chiba, Japan). A missense mutation in exon 2 of the CTLA4 gene (c.251T>C, p.V84A) was found and the mutation was confirmed by Sanger sequencing of the patient’s buccal cell DNA.

PreviouslyPublished: N/A

Variant: NM_001037631.2:c.251T>C

ClinVarID: N/A

CAID: CA350138385

gnomAD: N/A

SupplementalData: N/A

Note: Not functionally tested using transendocytosis