Activated phosphoinositide 3-kinase δ syndrome caused by PIK3CD mutations: expanding the phenotype

Patient 1, a 4-year and 10-month old Chinese boy, was admitted to our hospital for hepatosplenomegaly for two years, short stature for one and half year, and cough and fever for more than one week. He is the third child from healthy nonconsanguineous parents without related familial history of genetic diseases, and no obvious abnormality was found at birth. This patient had repeated respiratory tract infections during the last 3 years, with a frequency of 1–2 times a month.

At presentation, he was febrile (38.9^oC), anemic appearance, short stature (102 cm, less than 3rd ), low weight (15 kg, less than3rd ). In addition, enlarged lymph nodes could be palpated under the jaw, neck and behind the ear. Blood results revealed a slightly elevated level of white blood cell (WBC) count (10.12 x 10⁹/L), a significantly decreased hemoglobin (84 g/L) and serum IgG level (0.08 g/L), as well as abnormal lymphocyte proportion in peripheral blood detected by flow cytometry (shown in Table 1). EBV and CMV DNA in peripheral blood were negative. Bone marrow biopsy showed granulocytic hyperplasia and small cell low pigment changes in mature erythrocytes. Chest computer tomography (CT) revealed bilateral pneumonia and partial consolidation. Abdominal CT showed an increase in the volume of liver and spleen, and multiple enlarged lymph nodes in the mesentery and retroperitoneum, with the diameters of about 4–15 mm, and no obvious abnormal density was found in the parenchyma.

After admission, a laparotomy revealed enlarged liver and spleen, as well as small intestinal mesenteric lymph nodes. Two pieces of tissues (0.8 x 0.6 x 0.5 cm, respectively) from the margins of liver and spleen, and three larger lymph nodes, were taken for pathological examinations. During exploratory laparotomy, a Meckel diverticulum was observed and removed from about 50 cm away from the ileocecal region, opposite to the mesentery of the small intestine, after obtaining the consent of his parents. The biopsy of the surgical specimen demonstrated that the mucosal lymphoid tissue proliferation and positive EBER (EBV-encoded RNAs) in situ hybridization in a few cells, indicating lymphatic hyperplasia possibly caused by EBV infection, with no evidence of malignancy.

One month after discharge, the patient presented with swelling of both knee joints, especially the left knee, affecting normal activities. Magnetic Resonance Imaging (MRI) showed abnormal signal of fluid accumulation focusing in the bilateral knee joint cavities and the left knee suprapatellar capsule, as well as slightly swelling muscles surrounding and thickening and strengthening of the synovial membrane in the left knee joint. The swelling relieved after taking naproxen 10 ~ 15 mg/(kg.d) orally twice for three days.

Patient 2, an 8.5-year old boy, presented our allergy clinic with multiple ecchymosis for half day. The patient had no abnormality at birth and was in normal physical condition. At the age of 2 months old, he had been diagnosed as thrombocytopenia. At 5 years, he underwent cervical lymphadenectomy, and lymph node biopsy demonstrated reactive hyperplasia. At 7 years old, he received colonoscopy and polypectomy due to abdominal pain and diarrhea due to intussusception caused by multiple ileocecal polyps (Fig. 1A, B and C).

At presentation, he had multiple ecchymosis on both lower and limbs, scattered petechiae on limbs and trunk, and enlarged superficial lymph nodes on both side of the neck, with the largest one of 2cm x 2cm. His height and weight were normal. Physical examination and imaging identified no convincing enlargement of liver and spleen. Blood tests showed a low blood platelet count 72 (10^9/L), a normal count of red blood cells 4.53(10^12/L) and a slightly elevated count of WBC (5.55 x 10⁹/L), the levels of serum IgA, IgG and IgM were slightly increased (gamma globulin infusion one week before this test), and abnormal lymphocyte proportion in peripheral blood detected by flow cytometry (Table 1). In addition, the serum detection of CMV IgM was positive, however, the concentrations of CMV and EBV DNA were less than 4.00E + 02 copies/ml and 2.00E + 03 copies/ml, respectively, excluding the infection of CMV and EBV. Although the patient was initially diagnosed with immune thrombocytopenia, his medical history was pointing to immunodeficiency disease, thus, peripheral blood was drawn from the patient and his parents for trios WES.

Patient 3, a 7-year and 2-month old boy, was referred to the pneumology department due to recurrent respiratory infections. He was the only child born to a nonconsanguineous parents. His mother had recurrent infection and allergic rhinitis during her childhood. The patient had a history of multiple recurrent infections that had started from one month after birth, including bronchopneumonia, otitis media, amygdalitis, conjunctivitis, EBV infection, skin infections (mural abscess and furunculosis) and recurrent purpura, and even septicopymeia. At the age of 6 years old, he had recurrent hematuria, and the renal function tests showed elevated serum creatinine and uric acid, proteinuria and erythrocyturia, and the diagnosis of acute nephritic syndrome was considered. His parents refused to have renal puncture. The other clinical laboratory examinations were shown in Table 1. An ultrasound scan of kidneys and bilateral ureters was negative. The lung CT scan revealed right lung pneumonia with alveolar consolidation of right upper lobe (Fig. 1H). Bronchoscope examination found bronchial mucosal hypertrophy with follicular hyperplasia, and stenosis of the basal segment bronchial opening in the lower lobe of the right lung (Fig. 1D and G). Pathological analysis of tissues taken from the bronchus showed partial tissues were significantly compressed and a large number of inflammatory cells, mainly lymphocytes and plasma cells, in the epithelium and stroma. The immunohistochemistry staining revealed positive for CD3 and CD20, scattered positive CD138 cells. In addition, autoimmune serology was positive for perinuclear ANCA (p-ANCA) (the titer was 128) and negative for cytoplasmic ANCA (c-ANCA) and proteinase 3, according o the clinical manifestations and positive p-ANCA, the diagnosis of ANCA-associated vasculitis (AAV) was considered.

In order to determine the cause of the patient’s recurrent sinopulmonary infections, hepatosplenomegaly, and/or mucosal follicular hyperplasia for a clear diagnosis, trios-WES was conducted. Bioinformatic analysis was performed to identify candidate variants according to filtering strategies based on population frequency, variant classification, and variant functional damaging prediction, including Polymorphism Phenotyping v2, Sorting Intolerant From Tolerant, and Combined Annotation Dependent Depletion. A heterozygous mutation (c.3061G > A, p.E1021K) was found in PIK3CD gene (NM_005026) in patient 1 and patient 2. Sanger sequencing showed that this mutation was de novo and their parents did not carry this mutation. Another heterozygous mutation (c.1574 A > G, p.E525G) was found in patient 3, and this mutation was inherited from his mother (Fig. 2A and B). These two variants are pathogenic according to ACMG guidelines for variant classification (PS1 + PM1 + PM2 + PM5 + PP3). The diagnosis of APDS was made based on the patients’ clinical presentations and genotypes.

Compared with the control sample, the number of lymphocytes in the mucosa of patients was significantly increased. The polyp-like protrusion of intestinal mucosa in patient 2 was taken. Biopsy showed obvious hyperplasia of mucosal lymphoid tissue, formation of lymphoid follicles and proliferative lesions caused by chronic stimulation of the intestine. A large number of inflammatory cells mainly composed of lymphocytes and plasma cells could be seen in the bronchial mucosa epithelium and stroma of patient 3, which suggests chronic inflammatory changes. CD3 and CD20 are expressed in the cell membrane of lymphocytes and where CD3 labeled T lymphocytes while CD20 labeled B lymphocytes (Fig. 3). Immunohistochemical results showed that the number of T and B lymphocytes in mucosal tissue was significantly increased compared with control group. In addition, we found that the expression level of AKT in patients’ tissues was significantly increased which prompted for activation of AKT signal pathway in tissues (Fig. 3).