Case#: Case 1, female, Italian

DiseaseAssertion: APDS1

FamilyInfo: non-consanguineous parents

CaseHPOFreeText: Her clinical history included upper respiratory tract infections during adolescence and an episode of hidradenitis suppurativa at the age of 21 years. Family history was negative for primary immunodeficiencies. Immunological work up showed lymphopenia with hypogammaglobulinemia of all classes and absent response to vaccinations (Supplementary Table 1). T cells were present at normal percentages, while B cells were slightly below the lower range of the norm (Supplementary Table 1). The patient was diagnosed with CVID at 27 years of age and was started on immunoglobulin replacement treatment. During 20 years of follow-up, the clinical course of the patient was particularly mild: she only presented one episode of gastroenteritis at the age of 36 years, and occasional upper respiratory tract infections were treated with oral antibiotics. She has always remained negative for CMV and EBV. Annual abdominal ultrasonography showed a normal spleen size and no lymphoadenopathies. Lung CT scanning did not reveal mediastinal lymphoadenopathies or development of bronchiectasis during sequential lung CT scans (Supplementary Fig. 1). Endoscopic evaluation at 39 years revealed mild gastritis and duodenitis without T cell infiltrate with complete lack of plasma cells (Supplementary Fig. 2). Regarding the immunological evolution during follow-up, lymphopenia was persistent over time (Fig. 1A). In addition, the patient showed a progressive reduction of peripheral B cells (Fig. 1A and Supplementary Table 1) with lack of terminal B cell differentiation (Supplementary Table 2). Bone marrow evaluation showed impaired B cell maturation with an accumulation of precursors at the pro-B to pre-B1 stage (Fig. 1B).

CasePreviousTesting: NGS

GenotypingMethod: NGS followed by Sanger

Variant: c.1973C>T; p.P658L mutation in p110δ

CAID: CA577395

gnomAD: allele frequency of 0.0001114 (2/17954 alleles) in the East Asian population in gnomAD v2.1.1

Case#: Case 1, male

DiseaseAssertion: APDS

FamilyInfo: non-related Caucasian parents

CaseHPOFreeText: Suffered from Haemophilus b epiglottitis at the age of 2. He had received only one vaccine dose against diphtheria, tetanus, and poliomyelitis, due to parental choice, and had a history of recurrent respiratory tract infections. Biological features at diagnosis included: reduced serum levels of IgG2 and IgG4, normal IgA, IgG1, and IgG3 levels, and elevated IgM levels. The total lymphocyte count was normal but with quantitatively decreased T cells and CD21+ B cells, and an immune profile in favor of excessive memory CD4+ T cells. Later on, he suffered from frequent respiratory tract infections and a chest computed-tomography showed bronchiectasis at the age of 4. Digestive symptoms also appeared at the age of 4 when he presented with hematochezia related to colic malacoplakia (polypoid mucosal infiltration with histiocytes containing intra-cytoplasmic inclusions stained by Michaelis-Gutmann coloration) and lymphoid hyperplasia, which were both diagnosed on gastro-intestinal biopsies. From the age of 8 onward, he began to experience diarrhea that was linked to infections by Giardia and Cryptosporidium (diagnosed through acid-fast staining performed on stool samples). The cryptosporidiosis evolved toward a chronic infection with multiple episodic recurrences. He then developed celiac-mesenteric and hepatic lymphadenopathy, chronic ileitis with malabsorption syndrome, colitis with exudative diarrhea, and cholestasis with mild hepatic cytolysis due to grade II hepatic fibrosis (chronic hepatitis with inflammation and portal fibrosis, Metavir scoring F2-F3) with no sclerotic cholangitis. A recurrence of cryptosporidiosis accompanied by a C. difficile infection led to another intensive care unit stay, at the age of 9. Lymphadenopathy increased thereafter, with the appearance of hepatosplenomegaly, but lymphoma was not diagnosed on biopsies. He also developed cutaneous candidiasis, asymptomatic EBV reactivation (age 10) and persistent shedding of Adenovirus in the stools without viremia. The biological phenotype also worsened with time, leading to a TlowBlowNK+ CID. The evolution of the main immunologic parameters is shown in Fig. 1. Further analyses identified the following: absence of class-switched B cells, low and temporary immunoglobulin response to tetanus and diphtheria antigens and no response to Pneumococcus or Haemophilus b antigens, no lymphocyte proliferation to antigens after revaccination, and low or nonexistent proliferation with mitogens. Immunological explorations performed up to the age of 9 did not provide us with the precise diagnosis: IL-6 and IL-10 levels, double negative T cells, ADA and PNP levels, class I and II HLA molecules and CD40L, sequencing of CD40L and RAG1/2 genes, and Vβ repertoire of T cells were all normal. Proliferation of B cells with CD40L and IL-4 was present but weak.

CasePreviousTesting: No previous testing

GenotypingMethod: we decided to sequence PIK3CD in our patient. Sanger?

Variant: the E1021K mutation was identified.

CAID: CA145460

gnomAD: Absent from gnonAD v2.1.1

Case#: Case 1, male

DiseaseAssertion: APDS

FamilyInfo: consanguineous Pakistani parents. During the preparation of this manuscript, two kindreds with loss-of-function (LOF) variants in PIK3CD were published, all of whom had hypogammaglobulinemia and recurrent sinopulmonary infections (Table E1).3,4

CaseHPOFreeText: The patient received the attenuated Bacillus Calmette-Guérin, polio, and measles vaccines without sequelae and was healthy until six years of age, when he presented with chronic diarrhea and polyarticular arthritis affecting his knees and ankles. He was empirically treated with steroids for two months, leading to complete resolution of his symptoms. Over the subsequent five years, he had episodes of colitis treated with prednisone, sulfasalazine, and methotrexate for presumed inflammatory bowel disease. Due to worsening colitis, he underwent an upper endoscopy and colonoscopy that revealed candida esophagitis as well as increased intraepithelial lymphocytes and moderate villous blunting in the duodenum. His laboratory evaluation was notable for leukocytosis, neutrophilia, mild monocytosis, and thrombocytosis (Table 1). He had normal numbers of T, B, and NK cells and normal percentages of T and B cell subsets. His serum levels of IgG and IgA were decreased. Ca2+ flux in response to anti-CD3 crosslinking on T cells was decreased (Fig. 1A), although proliferation to anti-CD3+CD28 stimulation was robust (Table 1). The patient was treated with immunoglobulin replacement therapy, antifungal prophylaxis, and prophylactic antibiotics, but died at the age of 14 years due to a severe pneumonia and sepsis shortly after the mutation was identified.

CasePreviousTesting: WES

GenotypingMethod: WES

Variant: Whole-exome sequencing of the patient revealed a novel homozygous frameshift mutation in PIK3CD (c.2558_2559delAT; p.Asp853Glyfs*20), disrupting exons 20 – 24 encoding 171 amino acids of the ATP binding site within the catalytic domain

CAID: CA2499214908

gnomAD: absent from gnomAD v2.1.1

SupplementalData:

Case#: Patient 3, Korean, 13 year old, female

DiseaseAssertion: APDS1

CaseHPOFreeText: Previously diagnosed with selective IgA deficiency was reevaluated for other PIDs when she presented with multiple episodes of prolonged fever and enlargement of several lymph nodes. Patient 3 had a history of hospitalizations attributable to recurrent respiratory tract infections, cellulitis, and osteomyelitis. She was diagnosed with selective IgA deficiency at the age of 4 years at our hospital. Cervical lymph node enlargement was first noticed at the age of 6 years; hematochezia was also noted. Colon and cervical lymph node biopsies showed atypical lymphocyte proliferation with polyclonality, and persistent atypical lymphoid proliferation of the colon was observed at the age of 10 years. One colon biopsy specimen was suspicious for mucosa-associated lymphoid tissue lymphoma. The oncologists decided to observe the patient without special chemotherapy. During the follow-up period, we diagnosed and treated Patient 1 from the present case series, leading the clinicians to reevaluate Patient 3 for other PIDs. Upon reevaluation, the height and weight of Patient 3 were below the 5th percentile and development was normal. At the age of 12 years, laboratory values were WBC, 5410/µL; Hb, 8.5 g/dL; and PLT, 285k/µL. Immunological values were CD3, 391/µL (normal, 800–3500/µL); CD4, 194/µL (normal, 400–2100/µL); CD8, 190/µL (normal, 200–1200/µL); and CD19, 54/µL (normal, 200–600/µL). The CD4:CD8 ratio was 1.02. The response to phytohemagglutinin mitogen was reduced, while results from the same test performed 8 years prior were normal. Serum IgG, A, and M levels were 1920 mg/dL (normal, 639–1349 mg/dL), 3 mg/dL (normal, 70–312 mg/dL), and 1138 mg/dL (normal, 56–352 mg/dL), respectively. Hypermetabolic enlarged lymph nodes, splenomegaly, and bronchiectatic changes in the middle lobe of the right lung were observed on PET/CT. Summary of symptoms in Table 1.

CasePreviousTesting: exome sequencing

GenotypingMethod: Diagnostic exome sequencing confirmed by Sanger

Variant: E1021K, heterozygous

CAID: CA145460

gnomAD: variant is absent in gnomAD v2.1.1

Case#: Patient 2, Korean, 4 year old, male

DiseaseAssertion: APDS1

CaseHPOFreeText: history of idiopathic thrombocytopenic purpura, Bacillus Calmette-Guérin vaccine site infection, and respiratory tract infection requiring hospitalization was referred to our clinic for enlargement of multiple lymph nodes suspicious for lymphoma. Cervical lymph node enlargement was first noticed at 31 months old. At the age of 3 years, decreased IgG (89 mg/dL) with elevated IgM (347 mg/dL) and normal IgA (16 mg/dL) were detected at a prior hospital. He received regular IGRT under the interim diagnosis of CVID; however, trough IgG levels remained below 400 mg/dL despite receiving a higher IGRT dose (800 mg/kg) every 3 weeks. Patient 2 had a head circumference of 52.8 cm (95th percentile) and body weight of 17 kg (25–50th percentile). Multiple enlarged lymph nodes in the cervical, supraclavicular, and inguinal areas were palpated, and massive splenomegaly was noted. Molloscum contagiosum was observed on his buttocks. Complete blood count results included WBC, 3300/µL; Hb, 13.1 g/dL; and PLT, 157k/µL. Immunological values were CD3, 867/µL (normal, 1578–3707/µL); CD4, 241/µL (normal, 870–2144/µL); CD8, 613/µL (normal, 472–1107/µL); and CD19, 24/µL (normal, 276–640/µL). The CD4:CD8 ratio was 0.39 (Table 1). Positron emission tomography/computed tomography (PET/CT) findings showed enlargement of multiple lymph nodes, including the bilateral cervical, supraclavicular, and inguinal areas, and abnormal uptake in the whole intestine. A colonoscopy revealed multiple nodules and masses throughout the entire length of the colon. Cervical lymph node and large intestine biopsies revealed atypical lymphocyte proliferation with polyclonality (Fig. 1A–C) (Supplementary Fig. 1A and B, only online). A colon biopsy revealed CMV, and the patient complained of diarrhea. Summary of symptoms in Table 1.

CasePreviousTesting: exome sequencing

GenotypingMethod: Diagnostic exome sequencing confirmed by Sanger

Variant: E1021K, heterozygous

CAID: CA145460

gnomAD: variant is absent in gnomAD v2.1.1

Case#: Patient 1, Korean, 4 year old, male

DiseaseAssertion: APDS1

CaseHPOFreeText: diagnosed with common variable immunodeficiency (CVID) was referred to our clinic for persistent lymph node enlargement, hematochezia, and increased serum immunoglobulin (Ig)M. His medical history included double barrel enterostomy and segmental resection of the small intestine, attributable to intestinal malrotation and perforation in the neonatal period. He was diagnosed with hemolytic anemia at the age of 10 months and pancytopenia at the age of 15 months, after which he received steroid treatment. At the age of 21 months, he exhibited increased IgM (304 mg/dL; normal, 43–173 mg/dL) and decreased IgG (16 mg/dL; normal, 345–1236 mg/dL) and IgA (<1 mg/dL; normal, 11–106 mg/dL). Regular immunoglobulin-replacement treatment (IGRT, 500 mg/kg every 3 weeks) was initiated for CVID. At the age of 10 months, a lymph node biopsy revealed atypical lymphoid cell proliferation. At the age of 3 years, hematochezia gradually increased, and IgG trough levels were not well maintained (average, 333 mg/dL; normal, 345–1236 mg/dL) despite IGRT (500 mg/kg every 3 weeks). Patient 1 had a head circumference of 55 cm (>95th percentile) and body weight of 15 kg (<10th percentile), as well as multiple enlarged cervical lymph nodes and splenomegaly. Complete blood count values were white blood cells (WBC), 7910/µL (normal, 6000–15000/µL); hemoglobin (Hb), 10.9 g/dL (normal, 10.5–14.0 g/dL); and platelets (PLT), 122 k/µL (normal, 150–450 k/µL). Immunological values were CD3, 68%, 2320/µL (normal: 56–75%, 1400–3700/µL); CD4, 19%, 648/µL (normal: 28–47%, 700–2200/µL); CD8, 45%, 1535/µL (normal: 16–30%, 490–1300/µL); and CD19, 10%, 341/µL (normal: 8–39%, 180–1300/µL). Serum IgG, A, and M levels were 332 mg/dL (normal, 345–1236 mg/dL), <1 mg/dL (normal, 14–159 mg/dL), and 569 mg/dL (normal, 43–207 mg/dL), respectively (Table 1). Mucosal nodular lymphoid hyperplasia visualized as cobblestone-like polyps and cytomegalovirus (CMV) was detected in a mucosal biopsy through colonoscopy. Summary of symptoms in Table 1.

CasePreviousTesting: exome sequencing

GenotypingMethod: Diagnostic exome sequencing confirmed by Sanger

Variant: E1021K, heterozygous

CAID: CA145460

gnomAD: variant is absent in gnomAD v2.1.1

Case#: Wang_2018_P1, M, 2 y.o. (onset), origin in China

DiseaseAssertion: APDS

FamilyInfo: None reported

CasePresentingHPOs: RRTI (HP:0002205) Tonsillitis (HP:0011110) Diarrhea (HP:0002014) Lymphadenopathy (HP:0002716) Hepatomegaly (HP:0002240) Splenomegaly (HP:0001744) ASD (HP:0001631) Dacrocystitis (HP:0000620) Inguinal hernia (HP:0000023) autoimmune cytopenia (HP:0001973) increased transitional B cells (HP:0030381) increased plasmablasts (HP:0032128) decreased CD3 (HP:0045080) increased CD4 (HP:0032219) decreased CD4 naive (HP:0410378) increased CD4 EM (HP:0025625) decreased CD8 naive (HP:0410377) decreased T cells (HP:0005403) decreased b cells (HP:0010976) decreased CD4/CD8 (HP:0033222) decreased IgG (HP:0004315) increased IgM (HP:0003496)

HP:0002205, HP:0011110, HP:0002014, HP:0002716, HP:0002240, HP:0001744, HP:0001631, HP:0000620, HP:0000023, HP:0001973, HP:0030381, HP:0032128, HP:0045080, HP:0032219, HP:0410378, HP:0025625, HP:0410377, HP:0010976, HP:0004315, HP:0003496

CaseHPOFreeText: EBV DNA, decreased antibodies to Hepatitis-B, presence of autoantibodies, decreased CD19, increased CD8, increased CD8 CM, increased NK cells, increased CD8 EM

CaseNotHPOs: abnormal naive B cells (HP:0030370) abnormal memory B cells (HP:0030373) abnormal CD8 TEMRA (HP:0020177) abnormal wbc counts (HP:0011893) abnormal IgA (HP:0410240)

HP:0030370, HP:0020177, HP:0410240, HP:0011893, HP:0020177

CaseNotHPOFreeText: CMV-IgM negative, Fungus negative, abnormal CD4 CM, abnormal DNT

CasePreviousTesting: None reported

GenotypingMethod: WES + Sanger

PreviouslyPublished: Additional info published in 2022 (PMID:35799777)

Variant: NM_005026.5(PIK3CD):c.3061G>A (p.Glu1021Lys)

ClinVarID: 88675

CAID: CA145460

gnomAD: Not present in gnomAD

SupplementalData: Phenotypic info in table S4