Phosphatase and tensin homolog (PTEN) mutation can cause activated phosphatidylinositol 3-kinase δ syndrome–like immunodeficiency

Phosphatase and tensin homolog (PTEN) mutation can cause activated phosphatidylinositol 3-kinase d syndrome–like immunodeficiency Yuki Tsujita, MD,a* Kanako Mitsui-Sekinaka, MD,a* Kohsuke Imai, MD, PhD,a,b Tzu-Wen Yeh, MSc,c Noriko Mitsuiki, MD, PhD,c Takaki Asano, MD,d Hidenori Ohnishi, MD, PhD,e Zenichiro Kato, MD, PhD,e,f Yujin Sekinaka, MD,a Kiyotaka Zaha, MD,a Tamaki Kato, MD, PhD,a Tsubasa Okano, MD,c Takehiro Takashima, MD,c Kaoru Kobayashi, MD, PhD,g Mitsuaki Kimura, MD, PhD,h Tomoaki Kunitsu, MD,i Yoshihiro Maruo, MD, PhD,i Hirokazu Kanegane, MD, PhD,c Masatoshi Takagi, MD, PhD,c Kenichi Yoshida, MD, PhD,j Yusuke Okuno, MD, PhD,k Hideki Muramatsu, MD, PhD,k Yuichi Shiraishi, PhD,l Kenichi Chiba, BA,l Hiroko Tanaka, BSc,m Satoru Miyano, PhD,l,m Seiji Kojima, MD, PhD,k Seishi Ogawa, MD, PhD,j Osamu Ohara, PhD,n Satoshi Okada, MD, PhD,d Masao Kobayashi, MD, PhD,d Tomohiro Morio, MD, PhD,c and Shigeaki Nonoyama, MD, PhDa Saitama, Tokyo, Hiroshima, Gifu, Kobe, Shizuoka, Shiga, Kyoto, Nagoya, and Chiba, Japan GRAPHICAL ABSTRACT

PI3K

APDS1 p110δ-GOF APDS1 p85α-LOF APDS2

PIP2

PIP3

PTEN PHTS Macrocephaly

Hamartoma Ha amartoma

Tumor

APDS-L A PDS L

+ Immunodeficiency APDS: Ac vated PI3Kinase-Delta Syndrome APDS-L: APDS-like immunodeficiency PHTS: PTEN Hamartoma Tumor Syndrome

From athe Department of Pediatrics, National Defense Medical College, Saitama; the Departments of bCommunity Pediatrics, Perinatal and Maternal Medicine and cPediatrics and Developmental Biology, Tokyo Medical and Dental University (TMDU); dthe Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences; ethe Department of Pediatrics, Gifu University Graduate School of Medicine; fStructural Medicine, United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University; gthe Department of Surgery, Kuma Hospital, Kobe; hthe Department of Allergy and Clinical Immunology, Shizuoka Children’s Hospital; ithe Department of Pediatrics, Shiga University of Medical Science; jthe Department of Pathology and Tumor Biology, Kyoto University; kthe Department of Pediatrics, Nagoya University Gradual School of Medicine; lthe Laboratory of DNA Information Analysis and mthe Laboratory of Sequence Analysis, Human Genome Center, Institute of Medical Science University of Tokyo; and nthe Department of Technology Development, Kazusa DNA Research Institute, Chiba. *These authors contributed equally to this work. Supported in part by the Ministry of Defense, Japan; the Research on Measures for Intractable Diseases Project (H26-037, H23-012): matching fund subsidy from the Ministry of Health, Labour and Welfare, Japan; the Ministry of Education, Culture, Sports,

GOF (gain of func on) muta on LOF ( loss of func on) muta on

Science and Technology (MEXT), Japan (26293250, 15K09640); the Practical Research for Rare/Intractable Diseases from Japan Agency for Medical Research and Development, AMED; the Japan Foundation for Pediatric Research; the Jeffrey Modell Foundation; the Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics; and Drs M. and W. Hirose, H. Matsuda, and H. Seto. Disclosure of potential conflict of interest: K. Imai has received research support from the Japan Foundation for Pediatric Research and CSL Behring KK and has received consultancy and lecture fees from CSL Behring. The rest of the authors declare that they have no relevant conflicts of interest. Received for publication April 29, 2015; revised February 27, 2016; accepted for publication March 16, 2016. Corresponding author: Kohsuke Imai, MD, PhD, Department of Community Pediatrics, Perinatal and Maternal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. E-mail: [email protected]. 0091-6749/$36.00 Ó 2016 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaci.2016.03.055

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Background: Activated phosphatidylinositol 3-kinase d syndrome (APDS) is a recently discovered primary immunodeficiency disease (PID). Excess phosphatidylinositol 3kinase (PI3K) activity linked to mutations in 2 PI3K genes, PIK3CD and PIK3R1, causes APDS through hyperphosphorylation of AKT, mammalian target of rapamycin (mTOR), and S6. Objective: This study aimed to identify novel genes responsible for APDS. Methods: Whole-exome sequencing was performed in Japanese patients with PIDs. Immunophenotype was assessed through flow cytometry. Hyperphosphorylation of AKT, mTOR, and S6 in lymphocytes was examined through immunoblotting, flow cytometry, and multiplex assays. Results: We identified heterozygous mutations of phosphatase and tensin homolog (PTEN) in patients with PIDs. Immunoblotting and quantitative PCR analyses indicated that PTEN expression was decreased in these patients. Patients with PTEN mutations and those with PIK3CD mutations, including a novel E525A mutation, were further analyzed. The clinical symptoms and immunologic defects of patients with PTEN mutations, including lymphocytic AKT, mTOR, and S6 hyperphosphorylation, resemble those of patients with APDS. Because PTEN is known to suppress the PI3K pathway, it is likely that defective PTEN results in activation of the PI3K pathway. Conclusion: PTEN loss-of-function mutations can cause APDSlike immunodeficiency because of aberrant PI3K pathway activation in lymphocytes. (J Allergy Clin Immunol 2016;nnn:nnn-nnn.) Key words: Activated phosphatidylinositol 3-kinase d syndrome, catalytic subunit p110d of phosphatidylinositol 3-kinase, phosphatase and tensin homolog, primary immunodeficiency disease

Activated phosphatidylinositol 3-kinase d syndrome (APDS) is a primary immunodeficiency disease (PID) characterized by recurrent lower respiratory tract infections, hepatosplenomegaly, polyadenopathy, CD4 lymphopenia, and decreased classswitched memory B-cell counts.1,2 Autosomal dominant gainof-function (GOF) mutations in PIK3CD, encoding the catalytic subunit p110d of phosphatidylinositol 3-kinase (PI3K), are known to cause APDS, hyper-IgM syndrome, and primary sclerosing cholangitis3 and are associated with host susceptibility to B-cell malignancies.4,5 This manifestation of APDS is designated in this article as APDS1. An immunodeficiency similar to APDS1, designated in this article as APDS2, associated with mutations in PIK3R1 (encoding the p85a regulatory subunit of PI3K) was also recently reported.6,7 Increased PI3K activation associated with increased p110d activation has been observed in patients with APDS1 and those with APDS2. In lymphocytes the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway is critical for cell survival, proliferation, growth, and metabolism.8 The AKT and mTOR pathway is upregulated by receptor-mediated activation of PI3K, which results in phosphorylation of inactive phosphatidylinositol 3,4bisphosphate (PIP2) into active phosphatidylinositol 3,4,5triphosphate (PIP3). This inositol phospholipid contributes to the expression of various bioactive compounds by activating the AKT signaling pathway.

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Abbreviations used APDS: Activated phosphatidylinositol 3-kinase d syndrome BRRS: Bannayan-Riley-Ruvalcaba syndrome CS: Cowden syndrome CVID: Common variable immunodeficiency FITC: Fluorescein isothiocyanate GAPDH: Glyceraldehyde-3-phosphate dehydrogenase GOF: Gain of function IVIG: Intravenous immunoglobulin KREC: Kappa-deleting recombination excision circle LOF: Loss of function mTOR: Mammalian target of rapamycin NK: Natural killer pAKT: Phosphorylation of AKT at Ser473 PCP: Pneumocystis jiroveci pneumonia PET: Positron emission tomography PID: Primary immunodeficiency disease PI3K: Phosphatidylinositol 3-kinase PIP2: Phosphatidylinositol 3,4-bisphosphate PIP3: Phosphatidylinositol 3,4,5-triphosphate pmTOR: Phosphorylation of mTOR at Ser2448 pS6: Phosphorylation of ribosomal protein S6 at Ser235 and Ser236 PTEN: Phosphatase and tensin homolog qPCR: Real-time quantitative PCR ST: Sulfamethoxazole/trimethoprim TFH: Follicular helper T TrB: Transitional B TREC: T-cell receptor excision circle

Conversely, AKT signaling is downregulated through expression of phosphatase and tensin homolog (PTEN), which dephosphorylates PIP3 to PIP2 in human subjects, as well as in mouse models.9,10 Thus PTEN loss-of-function (LOF) mutations are understood to activate the AKT/mTOR pathway.11 Considering that an excess of PI3K activity caused by mutations in PIK3CD or PIK3R1 causes APDS1 or APDS2 in human subjects, PTEN dysfunction might cause an APDS-like syndrome by impairing PIP3 dephosphorylation, thereby activating AKT signaling. In the current study we systematically performed whole-exome sequencing for patients with PIDs and identified heterozygous PTEN mutations in 2 patients. Like patients carrying PIK3CD GOF (APDS1) and PIK3R1 LOF (APDS2) mutations, these 2 patients with PTEN mutations showed APDS-like clinical profiles. We propose that PTEN LOF mutations can cause increased AKT/mTOR/S6 signaling in lymphocytes and result in APDSlike immunodeficiency.

METHODS Cases Fig 1 shows the family trees of the patients in this study. P1, from kindred A, is a 3-year-old boy who was born at 40 weeks of gestational age to nonconsanguineous parents. At birth, P1 presented with meconium aspiration syndrome and a group B Streptococcus infection. He was treated with intravenous immunoglobulin (IVIG) and antibiotics. Pancytopenia was observed during the neonatal period but spontaneously resolved after 3 months. P1 was hospitalized with Pneumocystis jiroveci pneumonia (PCP) at 4 months of age, at which time hepatosplenomegaly was also observed. P1 had an inverted CD4/CD8 lymphocyte ratio (CD41, 33.1%; CD81, 37.3%) and low natural killer (NK) T-cell counts (0.010%). PHA-

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Kindred A PTEN (R233X) wt/wt

wt/wt

P1 wt/mut

Kindred B PTEN (R15fs) wt/wt

Kindred C PTEN (I5fs)

wt/wt

P2 wt/mut

P4 wt/mut

P3 wt/mut

Kindred D PIK3CD (E1021K) wt/wt

P5 wt/mut

wt/wt

Kindred E PIK3CD (E1021K) wt/wt

wt/wt

wt/wt

P6 wt/mut

Kindred F PIK3CD (E525A) wt/wt

P7 wt/mut

P8 wt/mut

P9 wt/mut

FIG 1. Family tree of patients given a diagnosis of PID. Black boxes and circles represent patients with identified or suspected disease-causing mutations. An identification number has been assigned to each patient (P1-P9).

induced blastogenesis was decreased to 13,700 ppm (normal, 20,50056,800 ppm). Serum IgM levels were increased (6.3 g/L) and serum IgG levels were high (13.42 g/L) at the age of 4 months. PCP was successfully treated with sulfamethoxazole/trimethoprim (ST). P1 was suspected to have severe combined immunodeficiency, and prophylactic treatment with itraconazole, ST, and acyclovir, as well as periodic IVIG therapy, was initiated. Genetic screening for severe combined immunodeficiency and X-linked lymphoproliferative syndrome–related genes was performed, but no mutation was identified in known responsible genes. At 5 months of age, P1 had a fever accompanied by pancytopenia, which resolved spontaneously. Bone marrow aspiration findings were normal, with no indication of myelodysplastic syndrome. Itraconazole was discontinued after confirming normal copy numbers of T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KRECs). However, despite continuing prophylaxis with ST, acyclovir, and IVIG, P1 had recurrent fever once a month. IVIG was discontinued at the age of 15 months, and his serum IgG level remained greater than 10 g/L. Positron emission tomography (PET) was performed when P1 was 2 years old to investigate the cause of recurrent fever, and systemic polylymphadenopathy was observed (Fig 2, A). P1 had received DPT vaccination 4 times during infancy, and antigen-specific antibody levels for pertussis were normal (agglutinin titer, 1:1280) at 2 years of age. An inverted CD4/CD8 lymphocyte ratio (CD41, 27.4%; CD81, 37.8%) and low NK T-cell counts (0.021%) were observed repeatedly. Moreover, a decrease in CD41 naive T-cell counts (133 cells/mL) was observed at 2 years of age. P1 presented with persistent polylymphadenopathy and hepatosplenomegaly at 3 years of age. He had macrocephaly and mental retardation but did not have hamartoma or malignancy. P2, from kindred B, is a 15-year-old girl who was born at 39 weeks of gestational age to nonconsanguineous parents. She had neonatal asphyxia at birth (5-minute Apgar score, 6) and was hospitalized for 3 weeks. She experienced recurrent lower respiratory tract infections starting at 2 months of age. Mild mental retardation was later observed. An adenoidectomy was performed when she was 4 years old. At 10 years of age, she had prolonged cough and fever and was given a diagnosis of pulmonary aspergillosis. At that

time, hypogammaglobulinemia (serum IgG, 1.71 g/L; IgA, 0.04 g/L; and IgM, 0.03 g/L) was observed, and P2 was started on monthly IVIG therapy. Despite normal B- and T-cell counts, fluorescence-activated cell sorting analysis revealed strikingly reduced numbers of memory B cells and class-switched memory (CD271IgM2IgD2) B cells; as such, P2 was given a diagnosis of common variable immunodeficiency (CVID). Kindred C was given a diagnosis of familial Cowden syndrome (CS) because of repeated occurrence of thyroid disease and intestinal polyposis within the family. P4 had thyroid nodules, intestinal polyposis, cutaneous facial papules, and acral keratosis at 26 years of age. Investigation of his family revealed that both his father and elder sister had undergone thyroid surgery, had intestinal polyposis, and had received a diagnosis of CS. P4’s father died of systemic metastasis of undifferentiated carcinoma of the thyroid. P3, P4’s son, had multiple nodules of the thyroid and intestinal polyposis at 10 years of age. Both P3 and P4 were given diagnoses of an adenomatous goiter and intestinal polyposis associated with CS, and total thyroidectomy was performed. PTEN mutation (c.13Adel, p.I5fsX) of P3 and P4 was identified by means of Sanger sequencing. Neither P3 nor P4 had a history suggestive of immunodeficiency. In addition, they had no immunologic deficits in lymphocyte numbers, lymphocyte subsets, serum immunoglobulin levels, or virus-specific antibody titers (see Table E1 in this article’s Online Repository at www.jacionline.org). Case reports and detailed material and methods for P7 to P9 (kindred F) with a novel PIK3CD mutation are described in the Methods section and Table E1 in this article’s Online Repository at www.jacionline.org.

Genetic analysis We studied 75 Japanese patients with PIDs using whole-exome sequencing with the Illumina HiSeq 2000 or HiSeq 1000, and exonic sequences were enriched by using the SureSelectXT Human All Exon V4 and A5 Kit (50 Mb; Agilent Technologies, Santa Clara, Calif) or the TruSeq Exome Enrichment Kit (Illumina, San Diego, Calif). Bioinformatic analysis was performed by using an in-house algorithm based on published tools.12,13 Identified single

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FIG 2. PET scanning of P1 and P6 after administration of the glucose analog 18F-fludeoxyglucose. A, Increased glucose uptake was observed in cervical and abdominal lymph nodes and in spleen tissue from P1. B, Increased glucose uptake was observed in cervical, abdominal, and axillary lymph nodes from P6.

nucleotide variants were filtered with dbSNP version 131, an in-house single nucleotide polymorphism database, and the Human Genetic Variation Database (see Fig E1 in this article’s Online Repository at www.jacionline.org). This analysis identified 4 patients with an E1021K mutation in PIK3CD (including P5 and P6) and also 2 patients (P1 and P2) with mutations in PTEN. We found 3 related patients (P7-P9) with an APDS-like phenotype and identified that they carried a novel heterozygous E525A mutation of PIK3CD by using a candidate gene approach. We also enrolled 2 patients (P3 and P4) with PTEN mutations and CS (who have previously been reported14,15). In total, we enrolled 9 patients in this study: 4 with PTEN, 2 with PIK3CD E1021K, and 3 with PIK3CD E525A mutations. All human subjects (or their guardians) provided written informed consent in accordance with the Helsinki principles for enrollment. The study was approved by the Institutional Review Board of the National Defense Medical College. As healthy controls, we obtained blood samples from healthy adults who provided informed consent.

Real-time PCR PTEN gene expression was measured by using real-time quantitative PCR (qPCR) with cDNA from whole blood samples as a template for TaqMan Gene Expression Assays specific for the PTEN gene (Hs02621230-s1; Life Technologies, Grand Island, NY). PTEN expression levels were normalized to b-actin (ACTB) gene expression levels (Hs01064291-g1, Life Technologies).

Cell culture PBMCs were isolated from whole blood by using Lymphoprep (1114545; Axis Shield, Dundee, Scotland), according to the manufacturer’s instructions. The cells were washed twice and resuspended at a density of 2 3 106 cells/mL in RPMI 1640 medium (22400-097; Gibco, Carlsbad, Calif) containing 10% FCS (16140-063, Gibco). PBMCs were then cultured with plate-bound antiCD3 and IL-2 (TLY Culture kit25: Lymphotec, Tokyo, Japan) for 48 hours, generating activated T lymphocytes.

Immunoblot analysis Cells were washed in RPMI medium and lysed for 30 minutes in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (1862209; Thermo Fisher Scientific, Waltham, Mass) and phosphatase inhibitor cocktail (390055; SERVA Electrophoresis GmbH, Heidelberg, Germany). The lysate was centrifuged at 15,000 rpm (15 minutes at 48C), and precipitated proteins were incubated at 988C for 5 minutes in Laemmli sample buffer (161-0737; Bio-Rad Laboratories, Hercules, Calif). Approximately 25mg aliquots of total protein were separated by means of SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and probed with anti-phospho-AKT (ser473; 4060; Cell Signaling Technology, Danvers, Mass), anti-AKT (4901; Cell Signaling Technology), anti–glyceraldehyde3-phosphate dehydrogenase (GAPDH; ab9485; Abcam, Cambridge, Mass), anti–phospho-S6 ribosomal protein (Ser235/236; 4875; Cell Signaling Technology), and anti-PTEN (9552; Cell Signaling Technology) antibodies.

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FIG 3. DNA electropherogram of the PTEN gene, PTEN gene expression, and abundance of PTEN protein. A, DNA electropherogram shows the position of the mutation in PTEN (arrow). B, PTEN gene expression in P1 versus a healthy control subject (n 5 1; error bars indicate SD of triplicate qPCR data), as normalized to ACTB gene expression levels. C, PTEN and GAPDH expression was measured by using immunoblotting in activated T-cell lysates from patients P1 to P4 and healthy control subjects.

Data were analyzed with a Student t test, Welch t test, or Pearson x2 test and are expressed as means 6 SEs or SDs.

whole-exome sequencing. These mutations in PTEN were validated by using Sanger sequencing (Fig 3, A). P1’s mutation (R233X) has been previously reported as a causative mutation for 3 patients with CS16 and a patient with Bannayan-RileyRuvalcaba syndrome (BRRS).17 Two related patients (P3 and P4) with CS had been given a diagnosis of carrying the heterozygous PTEN mutation c.13 del A (I5fsX18). This mutation was also validated by using Sanger sequencing (Fig 3, A). Two patients with a PIK3CD E1021K mutation (P5 and P6, Fig 1) were identified through whole-exome sequencing. This mutation was validated by using Sanger sequencing (data not shown). A novel heterozygous missense mutation, c. 1574 A>C (E525A), in the helical domain of PIK3CD was identified in 3 patients in one family (P7, 8, and 9; Fig 1 and see Fig E2, A, in this article’s Online Repository at www.jacionline.org). Functional prediction algorithms (SIFT and MutationTaster) and protein 3dimensional structure analysis (see Fig E2, B-D, as described in the Methods section in this article’s Online Repository) indicated that E525A is deleterious.

RESULTS Identification of PTEN and PIK3CD mutations Two patients (P1 and P2) with PTEN mutations, c.697 C>T (R233X) and c.41-42 insGA (R15fsX9), were identified through

Decreased PTEN mRNA and protein expression in patients with PTEN mutations The expression of PTEN mRNA in PBMCs was investigated by using qPCR and was found to be decreased in P1 relative to a

Multiplex assay Multiplex assay was performed with a MILLIPLEX MAP Kit (48-611MAG, 44-667MAG, 42-667MAG; Millipore, Billerica, Mass) on a Luminex 200 instrument (Luminex, Austin, Tex), according to the manufacturer’s protocol.

Phosflow analysis of AKT (Ser473) PBMCs were suspended at a density of 104 cells/mL in serum-free RPMI1640 medium (22400-097; Gibco) or in RPMI-1640 containing 10 mM of the p110d inhibitor IC87114 in the presence of anti-human CD3 (BD PharMingen, San Diego, Calif) or CD19 fluorescein isothiocyanate (FITC) antibody (BioLegend, San Diego, Calif) to detect AKT phosphorylation on Ser473. The cells were incubated for 20 minutes at 378C and washed twice and then permeabilized and stimulated for 10 minutes at 378C, fixed for 10 minutes at 378C with Cytofix buffer (554655; BD Biosciences, San Jose, Calif), and pelleted. Cells were permeabilized in PERM III buffer (558050; BD Biosciences) for 30 minutes on ice (protocol III), stained with FITC-conjugated anti-CD3 or CD19 and Alexa Fluor 647–conjugated anti-phospho AKT (Ser473; D9E; Cell Signaling Technology) antibodies, and subjected to flow cytometric analysis.

Statistical analysis

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FIG 4. Hyperphosphorylation of AKT, mTOR, and S6 ribosomal protein in patients and healthy control subjects. A, Immunoblot analysis of AKT phosphorylation at Ser473 (pAKT[S473]), total AKT, S6 ribosomal protein phosphorylated at Ser235 and Ser236 (pS6[S235,236]), and GAPDH (loading control) in activated T cells. B, Multiplex assays of phosphorylation levels of pAKT(S473)/GADPH, pmTOR (S2448)/GADPH, and pS6(S235, S236)/GADPH in activated T cells. Fig 4, A, shows representative data of P1 (PTEN), P5 (PIK3CD E1021K), and P9 (PIK3CD E525A) and their controls (n 5 1). In Fig 4, B, Data from patients with PTEN, PIK3CD E1021K, and PIK3CD E525A mutations are shown relative to those from healthy control subjects (n 5 3, 2, and 1, respectively). C, Hyperphosphorylation of AKT in patients and healthy control subjects. Phosphorylation of pAKT in B cells in the resting state shown by using Phosflow analysis (red solid lines) and phosphorylation of pAKT in B cells from patients and healthy control subjects treated with p110d inhibitor (black dotted lines).

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healthy control subject (Fig 3, B). PTEN protein expression in activated T cells was assessed by using immunoblot analysis. Compared with healthy control subjects, PTEN protein expression was only weakly observed in patients with PTEN mutations (P1-P4). The signal intensity of the PTEN band from P1 was reduced to 11.3% of control signal intensity (Fig 3, C). These results suggest that the PTEN R233X allele is a LOF mutation that results in the loss of PTEN mRNA and protein expression. Although PTEN mRNA levels in 3 patients with PTEN mutations (P2-P4) were comparable with those of healthy control subjects (data not shown), immunoblot analysis revealed that these patients’ PTEN protein levels were decreased to approximately 60% of those of healthy control subjects (Fig 3, C). Thus we concluded that all PTEN mutations (R233X, R15fsX, and I5fsX) included in our study were LOF mutations.

Aberrant activation of the AKT/mTOR/S6 pathway in patients with PTEN mutations Increased PI3K activity leads to aberrant activation of the AKT/ mTOR/S6 pathway and is thought to be a major molecular cause of APDS1 and APDS2.1,2,6 Indeed, these patients show excess phosphorylation of AKT at Ser473 (pAKT), mTOR at Ser2448 (pmTOR), and ribosomal protein S6 at Ser235 and 236 (pS6). First, we investigated the expression of pAKT and pS6 in activated T cells using immunoblot analysis (Fig 4, A). AKT protein expression in all patients was indistinguishable from that of healthy control subjects. However, pAKT expression was significantly increased in all samples from patients with PTEN mutations (P < .01; Fig 4, A). Activated T cells from patients with PIK3CD mutations (E1021K and E525A) also showed increased pAKT expression. These findings are consistent with previous research.1,2 pS6 expression was also increased in all patients with PTEN mutations (P1-P4) and in those with PIK3CD mutations (P5-P9), although this increase was not as pronounced as that observed with pAKT. We quantified these results using densitometry and reanalyzed them with a protein ratio. Patient levels of pAKT/ GAPDH, pAKT/AKT, and pS6/GAPDH were increased 1.44- to 7.86-fold, 1.42- to 4.86-fold, and 1.49- to 4.90-fold, respectively, when compared with those of healthy control subjects. Activated T cells were subjected to multiplex analysis to assess the expression of pAKT, pmTOR, and pS6. All patients showed increased pAKT, pmTOR, and pS6 expression when compared with healthy control subjects (Fig 4, B, and see Fig E3 in this article’s Online Repository at www.jacionline.org). These results suggest that the AKT/mTOR/S6 pathway is aberrantly activated in activated T cells of both patients with mutated PTEN and those with APDS. We also investigated pAKT expression in CD191 B cells from PBMCs using flow cytometry. Cells from patients with mutated PTEN showed increased pAKT expression relative to that in healthy control subjects (Fig 4, C, and see Fig E4 in this article’s Online Repository at www.jacionline.org). Similar results were observed in cells from patients with APDS with PIK3CD E1021K and E525A mutations when compared with those from healthy control subjects (Fig 4, C, and see Fig E4). This increase in pAKT expression was reversed by p110d inhibitor treatment. However, inhibition was not observed in cells from healthy control subjects (Fig 4, C, and see Fig E4). Collectively, these data indicate that heterozygous GOF mutations in PIK3CD and heterozygous LOF mutations in PTEN result in aberrant activation of the AKT/mTOR/S6 pathway.

TABLE I. Clinical and immunologic features of the patients

Recurrent infection Lymphadenopathy Hepatosplenomegaly Lymphoma Lymphopenia CD4/CD8 inversion Increased TFH cell numbers Increased TrB cell numbers

PTEN (P1 and P2), n 5 2

APDS (P5-P9), n 5 5

100%, 2/2 100%, 2/2 50%, 1/2 0%, 0/2 50%, 1/2 50%, 1/2 0%, 0/2 0%, 0/2

80%, 4/5 75%, 3/4 40%, 2/5 0%, 0/2 20%, 1/5 40%, 2/5 100%, 5/5 100%, 5/5

Follicular T-cell and transitional B-cell counts increase in patients with APDS but not in patients with mutated PTEN Follicular helper T (TFH) cells are a distinct subset of CD41 helper T cells that play a central role in the initiation of classical B-cell responses.18,19 Transitional B (TrB) cells are developmentally intermediate B cells between immature bone marrow B-lineage cells and fully mature naive B cells in the peripheral blood and secondary lymphoid tissues.20 PBMCs were separated, and CD41CD31 CD45RO1CXCR51 TFH cells and CD191CD2411CD3811 TrB cells were analyzed by using flow cytometry. Percentages of TFH cells in our patients with PIK3CD mutations, including P5 to P9, were significantly higher than those in patients with CVID (mean 6 SD: 16.6 6 7.26 vs 4.83 6 4.86; P < .0001). Percentages of TrB cells in patients with PIK3CD mutations, including P5 to P9, were also significantly higher than those in patients with CVID (mean 6 SD: 57.0 6 28.2 vs 9.59 6 7.15; P < .0001). In contrast, the percentages of TFH and TrB cells in patients with PTEN mutations were comparable with those in patients with CVID (see Fig E5 in this article’s Online Repository at www.jacionline.org).

Comparison of clinical and immunologic manifestations in patients with mutated PTEN and PIK3CD Clinical characteristics of patients with PTEN mutations and those with PIK3CD mutations are described in Table E1. Of the 4 patients with PTEN mutations, P1 and P2, who had immunodeficiency, exhibited clinical symptoms similar to those of patients with APDS (P5-P9). Table I presents shared and distinct clinical and immunologic features between immunodeficient patients with PTEN LOF mutations (P1 and P2) and patients with APDS (P5-P9). With regard to infection episodes, P1 and P2 had opportunistic infections (PCP and pulmonary aspergillosis, respectively). P7 and P8 exhibited recurrent lower respiratory tract infections or recurrent fever, as previously reported in patients with PIK3CD E1021K and E525K mutations.1,2 P9 had no remarkable infection episodes. Lymphopenia was observed in P1 and P6. An inverted CD4/ CD8 ratio was found in P1, P5, and P7. P1 also presented with transient pancytopenia accompanied by infections. Lymphocyte blastogenesis by PHA was decreased in P1 and in patients with APDS (P5-P9). Serum IgM levels in patients with PTEN mutations were variable, whereas those in patients with a PIK3CD E525A mutation were normal or increased. Although patients with PTEN mutations presented with normal serum IgG2 levels, patients

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with APDS (P5-P9) showed decreased serum IgG2 levels (see Table E1). Lymphadenopathy was observed both in patients with PTEN mutations and those with APDS (see Table E1). Briefly, with regard to patients with PTEN mutations, P1 presented with lymphadenopathy in the cervical and abdominal lymph nodes and hepatosplenomegaly, and P2 had an enlarged adenoid requiring adenoidectomy. Regarding patients with GOF PIK3CD mutations, P5 presented with airway obstruction caused by aggressive enlarged cervical lymphadenopathy. P6 presented with hyperplastic lymphoid follicles in the ileocolonic mucosa. P7 presented with cervical, mediastinal, and mesenteric lymphadenopathy and hepatosplenomegaly. P8 and P9 presented with cervical lymphadenopathy, for which biopsies were performed and revealed no malignancies. P9 was suspected to have a mucosa-associated lymphoid tissue lymphoma of the rectum at 39 years of age. Because PET analysis is a useful tool to detect lymphoproliferation through increased glucose uptake in patients with APDS,2 we performed PET in P1 and P6. As shown in Fig 2, A, increased glucose uptake was detected in the cervical and abdominal lymph nodes, as well as in the spleen, reflecting systemic lymphadenopathy. P1 and P6 also showed systemic lymphadenopathy that was mainly detected in the axillary and abdominal lymph nodes (P6; Fig 2, B). Macrocephaly and mental retardation were observed in P1 and P2, as is also observed in patients with PTEN hamartoma tumor syndrome, CS, and BRRS.19 Although macrocephaly and mental retardation have not been reported in patients with PIK3CD mutation, we found both in 1 patient with PIK3CD mutation (P5). Generally, patients with PTEN mutations presented similar immunologic profiles to those of patients with APDS.

DISCUSSION We found that 2 (P1 and P2) of 4 (P1, P2, P3, and P4) patients with heterozygous LOF mutations in PTEN presented with immunodeficiency. Clinical presentations of these 2 patients (P1 and P2) resemble an APDS phenotype. Both patients had recurrent infections and systemic lymphadenopathy. P1 had lymphopenia, an inverted CD4/CD8 ratio, and high serum IgM levels; P2 had hypogammaglobulinemia and had been given a diagnosis of CVID. We investigated the AKT/mTOR/S6 pathway status in lymphocytes of these 4 patients with PTEN LOF mutations. We found that patients with PTEN mutations showed increased phosphorylation of AKT, mTOR, and S6, indicating aberrant activation of the AKT/mTOR/S6 pathway similar to that observed in patients with APDS. APDS is caused by PIK3CD or PIK3R1 mutations that result in PI3K hyperactivity. Considering that PTEN converts PIP3 to PIP2 (in contrast to PI3K, which converts PIP2 to PIP3), it is plausible that PTEN LOF mutations can cause APDS-like immunodeficiency (Fig 5). In addition, DNA sequencing and whole-exome analysis revealed normal sequences for PIK3CD, PI3KR1, and other immune system–related genes in P1 and P2. These data suggest that the APDS-like immunodeficiency observed in P1 and P2 was caused by PTEN LOF mutations. Mutations in PTEN have been previously identified in patients with PTEN hamartoma tumor syndrome.21 In addition, 80% of patients with CS, 60% of those with BRRS, up to 20% of those with Proteus syndrome, and approximately 50% of those with

J ALLERGY CLIN IMMUNOL nnn 2016

p110δ

PI3K

PIP2

PIP3 PTEN

PDK1

P AKT P mTOR

p70S6 P RPS6 FIG 5. Schematic representation. GOF mutations in PIK3CD favor the conversion of PIP2 to PIP3 and enhance phosphorylation of AKT, mTOR, and S6 proteins. Similarly, loss of PTEN function suppresses the inhibitory function of PI3K, which enhances protein phosphorylation in the AKT/mTOR/S6 pathway.

Proteus-like syndrome have been reported to carry PTEN mutations.21 Although some patients with CS, BRRS, and Proteus syndrome have been reported to be immunodeficient,22-26 PTEN mutations have not been analyzed in these patients and have not been considered to cause immunodeficiency. However, a case report about patients with CS with combined immunodeficiency carrying PTEN mutations has recently been published.27 Such reports are consistent with our observation that PTEN LOF mutations can cause APDS-like immunodeficiency. The reason why immunodeficiency occurs only in some, but not all, patients with PTEN LOF mutations is currently unknown. There are at least 3 possibilities. First, incomplete penetrance might explain the lack of immunodeficiency in some patients with PTEN LOF mutations. Second, considering that P3 and P4 have no immunodeficiency and are in the same family with the same PTEN mutation, there might be genotype-phenotype correlations; that is, it might be that only specific PTEN mutations cause immunodeficiency. Third, there might be a separate signal transduction pathway associated with PTEN that causes immunodeficiency because all 4 patients (P1-P4) with PTEN LOF mutations regardless of presence or absence of immunodeficiency exhibit increased phosphorylation of AKT/mTOR/S6 in activated T cells and B lymphocytes. Our future studies will address the cause of immunodeficiency observed in patients with PTEN mutations. Further studies with careful clinical observations and detailed immunologic investigations of additional patients with PTEN LOF mutations will clarify the immunologic effects of PTEN mutations. We identified immunodeficient patients with heterozygous PTEN LOF mutations. In these patients’ lymphocytes AKT/ mTOR/S6 were hyperphosphorylated, as is observed in patients

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with APDS. We conclude that PTEN LOF mutations can cause APDS-like immunodeficiency. We thank Ms Kaori Tomita and Ms Kimiko Gasa for their skillful technical assistance, Dr Masaki Fuyama for providing the patient data for this study, and Ms Nicole Luche for her many helpful comments.

Key messages d

Patients with PIDs with PTEN LOF mutations were identified.

d

AKT, mTOR, and S6 were hyperphosphorylated in lymphocytes of these patients.

d

PTEN LOF mutations immunodeficiency.

can

cause

APDS-like

REFERENCES 1. Angulo I, Vadas O, Garc¸on F, Banham-Hall E, Plagnol V, Leahy TR, et al. Phosphoinositide 3-kinase d gene mutation predisposes to respiratory infection and airway damage. Science 2013;342:866-71. 2. Lucas CL, Kuehn HS, Zhao F, Niemela JE, Deenick EK, Palendira U, et al. Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110d result in T cell senescence and human immunodeficiency. Nat Immunol 2014;15:88-97. 3. Hartman HN, Niemela J, Hintermeyer MK, Garofalo M, Stoddard J, Verbsky JW, et al. Gain of function mutations of PIK3CD as a cause of primary sclerosing cholangitis. J Clin Immunol 2014;35:11-4. 4. Crank MC, Grossman JK, Moir S, Pittaluga S, Buckner CM, Kardava L, et al. Mutations in PIK3CD can cause hyper IgM syndrome (HIGM) associated with increased cancer susceptibility. J Clin Immunol 2014;34:272-6. 5. Kracker S, Curtis J, Ibrahim MAA, Sediva A, Salisbury J, Campr V, et al. Occurrence of B-cell lymphomas in patients with activated phosphoinositide 3-kinase d syndrome. J Allergy Clin Immunol 2014;134:233-6. 6. Deau M-C, Heurtier L, Frange P, Suarez F, Bole-Feysot C, Nitschke P, et al. A human immunodeficiency caused by mutations in the PIK3R1 gene. J Clin Invest 2014;124:3923-8. 7. Lucas CL, Zhang Y, Venida A, Wang Y, Hughes J, McElwee J, et al. Heterozygous splice mutation in PIK3R1 causes human immunodeficiency with lymphoproliferation due to dominant activation of PI3K. J Exp Med 2014;211: 2537-47. 8. Okkenhaug K, Vanhaesebroeck B. PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol 2003;3:317-30.

9. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998;273:13375-8. 10. Li DM, Sun H. PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells. Proc Natl Acad Sci U S A 1998;95: 15406-11. 11. Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998;95:29-39. 12. Kunishima S, Okuno Y, Yoshida K, Shiraishi Y, Sanada M, Muramatsu H, et al. ACTN1 mutations cause congenital macrothrombocytopenia. Am J Hum Genet 2013;92:431-8. 13. Oda H, Nakagawa K, Abe J, Awaya T, Funabiki M, Hijikata A, et al. AicardiGouti
eres syndrome is caused by IFIH1 mutations. Am J Hum Genet 2014;95: 121-5. 14. Kobayashi K, Ohno K, Fukata S, Yokozawa T, Hirai K, Matsuzuka F, et al. Total thyroidectomy for an adenomatous goiter associated with Cowden’s disease: a case report. Endocr Surg 2002;19:116-9. 15. Kobayashi K, Hirokawa M, Amino N, Miyauchi A, Kogai T, Hishinuma A. A case report of Cowden’s disease with a mutation in PTEN gene. J Jpn Thyroid Assoc 2013;4:50-2. 16. Liaw D, Marsh DJ, Li J, Dahia PL, Wang SI, Zheng Z, et al. Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet 1997;16:64-7. 17. Marsh DJ, Dahia PLM, Zheng Z, Liaw D, Parsons R, Gorlin RJ, et al. Germline mutations in PTEN are present in Bannayan-Zonana syndrome. Nat Genet 1997; 16:333-4. 18. Breitfeld D, Ohl L, Kremmer E, Ellwart J, Sallusto F, Lipp M, et al. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med 2000;192:1545-52. 19. Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B. CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J Exp Med 2000;192:1553-62. 20. Carsetti R, K€ohler G, Lamers MC. Transitional B cells are the target of negative selection in the B cell compartment. J Exp Med 1995;181:2129-40. 21. Eng C. PTEN: one gene, many syndromes. Hum Mutat 2003;22:183-98. 22. Ruschak PJ, Kauh YC, Luscombe HA. Cowden’s disease associated with immunodeficiency. Arch Dermatol 1981;117:573-5. 23. Riley HD Jr, Smith WR. Macrocephaly, pseudopapilledema and multiple hemangiomata. A previously undescribed heredofamilial syndrome. Pediatrics 1960;26:293-300. 24. Halevy S, Sandbank M, Pick AI, Feuerman EJ. Cowden’s disease in three siblings: electron-microscope and immunological studies. Acta Derm Venereol 1985;65:126-31. 25. Amer M, Mostafa FF, Attwa EM, Ibrahim S. Cowden’s syndrome: a clinical, immunological, and histopathological study. Int J Dermatol 2011;50:516-21. 26. Hodge D, Misbah SA, Mueller RF, Glass EJ, Chetcuti PA. Proteus syndrome and immunodeficiency. Arch Dis Child 2000;82:234-5. 27. Browning MJ, Chandra A, Carvonaro V, Okkenhaug K, Barwell J. Cowden’s syndrome with immunodeficiency. J Med Genet 2015;52:856-9.

9.e1 TSUJITA ET AL

METHODS Case report of patients with PIK3CD GOF mutations P5 and P6 present with a known E1021K mutation in p110d. The clinical manifestations are similar to those previously reported.E1,E2 P7 and P9 are siblings (kindred F) born to nonconsanguineous parents. P7 is a proband who has had recurrent lower respiratory tract infections and otitis media starting at 2 years of age. Multiple lymphadenopathies of the mediastina and mesenteric lymph nodes were also observed at age 2 years. A histopathologic analysis was not performed because consent for lymph node biopsy could not be obtained. Increased serum IgM and low serum IgG2 levels (2.55 and 0.012 g/L, respectively) were found at 2 years of age. The patient had cellulitis and a herpes zoster infection at 9 years of age. He was hospitalized for sepsis at 10 years of age. Immunologic examination revealed low CD41 Tcell, low CD41 naive T-cell, high CD81 T-cell, low memory B-cell, low NK cell, and low NK T-cell counts. TREC and KREC levels were normal in P7 at 11 years of age. A novel heterozygous mutation, E525A, in PIK3CD was detected by using a candidate gene approach at 11 years of age. P9 is P7’s sister. Despite having multiple lymphadenopathies, P9 has no history of recurrent fever or remarkable bacterial infection. The histopathologic findings of enlarged cervical lymph nodes showed phagocytosing macrophages without any finding of monoclonality. She presented with low CD41 naive T-cell, low memory B-cell, high NK cell, and low NK T-cell counts. TREC and KREC levels were within the normal range. The E525A mutation in PIK3CD identified in P7 was identified in P9 through a familial study at age 12 years. After identification of the PIK3CD mutation, low serum IgG2 levels (<0.66 g/L; normal 12-year-old, 1.8-8.7 g/L) were observed. P8 is P7’s mother. She experienced recurrent fever and multiple lymphadenopathies in childhood. Lymph node biopsy was performed and revealed no malignancy. She was suspected of having lymphoma of mucosa-associated lymphoid tissue of the rectum at 39 years of age and has been followed up with regular colonoscopies. The E525A PIK3CD mutation was identified in P8 through familial study at the age of 40 years. She then received immunologic examination and presented with low CD41 naive T-cell counts, low serum IgG2 levels (0.98 g/L; normal adult, 2.65-9.31 g/L), and undetectable TRECs.

Real-time PCR Expression levels of KRECs (coding joint KRECs; cjKRECs and signal joint KRECs; sjKRECs) and TRECs were determined by using qPCR with genomic DNA from whole blood samples and specific primers and probes, as previously reported.E3,E4

Measurement of TREC and KREC levels TREC and KREC levels were measured in genomic DNA from whole blood samples to assess T- and B-cell generation. Normal KREC levels were detected in all patients. In patients with mutated PTEN, TREC levels were normal. In contrast, some patients with APDS showed decreased levels of TRECs. Briefly, P5, P6, and P8 were classified as TREC2/KREC1 (Table E2 and Fig E6). Although P5 presented normal TREC levels at the age of

J ALLERGY CLIN IMMUNOL nnn 2016

5 years, these were decreased in a blood sample obtained at age 8 years (Fig E6, B). P7, P8, and P9 are familial cases carrying the same PIK3CD E525A mutation. However, only P8 in this family, the oldest patient, had decreased TREC levels (Fig E6, C). Thus we suspect that patients with APDS present with an age-dependent reduction in TREC levels.

Flow cytometry Lymphocyte subset analysis was performed on whole blood after cell lysis by using optimized FITC-, phycoerythrin-, PC5-, peridinin-chlorophyllprotein complex–, and allophycocyanin-labeled combinations of antibodies: anti-CD3 (349201 [BD Biosciences] or IM2467 [Beckman Coulter, Miami, Fla]), anti-CD4 (347413 or 347324; BD Biosciences), anti-CD8 (340046; BD Biosciences), anti-CD16 (347617; BD Biosciences), anti-CD19 (IM2470; Beckman Coulter), anti-CD21 (PN IM473U; Beckman Coulter), anti-CD24 (PN OM1428U; Beckman Coulter), anti-CD25 (347643; BD Biosciences), anti-CD27 (PN IM2578; Beckman Coulter), anti-CD38 (340439; BD Biosciences), anti-CD56 (AO4489; Beckman Coulter), anti-CD127 (12-1278-73; BD Biosciences), anti-CD45RA (347723; BD Biosciences), anti-CD45RO (340438; BD Biosciences), anti–T-cell receptor Va24 (PN IM1589; Beckman Coulter), anti–T-cell receptor Vb11 (PN IM2290; Beckman Coulter), anti-IgD (55778; BD Biosciences), and anti-IgM (551079; BD Biosciences).

Structural analysis of PIK3 p110d mutations The human PI3K p110d protein subunit with p85-nSH2 and p85-iSH2 was modeled by using the p110a/p85a complex structure (PDB:3HHM) as a template on MOE software.E5,E6 Mutation-related effects were analyzed by creating the mutated protein structures.

REFERENCES E1. Angulo I, Vadas O, Garc¸on F, Banham-Hall E, Plagnol V, Leahy TR, et al. Phosphoinositide 3-kinase d gene mutation predisposes to respiratory infection and airway damage. Science 2013;342:866-71. E2. Lucas CL, Kuehn HS, Zhao F, Niemela JE, Deenick EK, Palendira U, et al. Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110d result in T cell senescence and human immunodeficiency. Nat Immunol 2014;15:88-97. E3. Morinishi Y, Imai K, Nakagawa N, Sato H, Horiuchi K, Ohtsuka Y, et al. Identification of severe combined immunodeficiency by T-cell receptor excision circles quantification using neonatal Guthrie cards. J Pediatr 2009;155:829-33. E4. Nakagawa N, Imai K, Kanegane H, Sato H, Yamada M, Kondoh K, et al. Quantification of k-deleting recombination excision circles in Guthrie cards for the identification of early B-cell maturation defects. J Allergy Clin Immunol 2011;128: 223-5. E5. Levitt M. Accurate modeling of protein conformation by automatic segment matching. J Mol Biol 1992;226:507-33. E6. Fechteler T, Dengler U, Schomburg D. Prediction of protein three-dimensional structures in insertion and deletion regions: a procedure for searching data bases of representative protein fragments using geometric scoring criteria. J Mol Biol 1995;253:114-31.

TSUJITA ET AL 9.e2

J ALLERGY CLIN IMMUNOL VOLUME nnn, NUMBER nn

Exome sequencing and filter procedure Single NucleoƟde and InserƟon-DeleƟon Variants 53210

Nonsynonymous SNVs and Indels 21917

Role of Rare Variants 10606

Ranking by allele frequency 242 De novo(3), Compound Heterozygous(0), and Hemizygous(3) variants

Candidate Varitans including known PTEN mutaƟon 3 (PTEN, B3GALT2, PIEZO2)

Filter 1 Exclude synonymous and unknown-funcƟonal variants

Filter 2 Exclude SNPs in dbSNP131 and inhouseSNP Filter3 Exclude SNVs and indels having allele frequencies of less than 0.25 (SNVs) and 0.10 (indels) Filter 4 Exclude variants shared by the each parents Filter 5 Exclude Japanese SNPs 0 GeneƟc in Human Variant Browser

FIG E1. Analysis algorithm for whole-exome sequencing data and location of the pathogenic PTEN mutation in P1. A data analysis algorithm was used to filter all single nucleotide variants and insertion-deletion variants. Numbers of variants identified by using exome-wide sequencing are shown for each filtering step. Although variations in 2 other genes (B3GALT2 and PIEZO2) were also annotated by using whole-exome sequencing, they were not considered to be involved in the disease because both were missense variations and the mutated genes were not related to the immune system. SNP, Single nucleotide polymorphism.

9.e3 TSUJITA ET AL

J ALLERGY CLIN IMMUNOL nnn 2016

FIG E2. DNA electropherograms of PIK3CD and a structural model of human PI3K. A, Novel mutations in the PIK3CD gene were identified in 3 related patients (P7-P9). DNA electropherograms show the position of the mutation (arrow). B and C, Normal structure (E525) showing interactions between p110d (blue) and the inter-SH2 (iSH2) and C-terminal SH2 (cSH2) domains of p85a (orange). D, Reported mutation (E525K). E, Novel mutation (E525A). Positive and negative charges are shown with red and blue sticks, respectively. Functional prediction algorithms (SIFT and MutationTaster) indicated that the novel E525A mutation might be deleterious. Moreover, in agreement with a previous report of the E525K mutation,E2 the new PIK3CD (E525A) mutation was associated with a loss of hydrogen bonds between the p85a and p110d subunits, resulting in decreased binding affinity. In addition, hydrogen bonds in the p110d molecule were lost, resulting in decreased stability of the peripheral p110d subunit, as indicated by using protein structure analysis. Mutations of A525 are less likely to affect steric hindrance because of the small size of the p110d subunit. However, binding affinity is decreased because of the loss of negatively charged residues (Fig E2, E). In contrast, mutations of K525 introduce positive charges that cause repulsive intermolecular forces (Fig E2, D). Thus both p110d mutations are expected to weaken binding to p85a and contribute to pathogenesis.

J ALLERGY CLIN IMMUNOL VOLUME nnn, NUMBER nn

FIG E3. Relative units of phosphorylation for AKT (Ser473), mTOR (Ser2448), and S6 (Ser235 and Ser236) by using multiplex assays of P2-P4 and P6-P8.

TSUJITA ET AL 9.e4

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J ALLERGY CLIN IMMUNOL nnn 2016

FIG E4. Hyperphosphorylation of AKT in P2-P4, P6-P8 and healthy control subjects.

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FIG E5. Representative flow cytometric analysis of TFH and TrB cells in patients with PIDs. A, Fluorescenceactivated cell sorting analysis of CD41CD31CD451CACR51 TFH cells of patients with PIDs with PIK3CD and PTEN mutations and patients with CVID. B, Representative flow cytometric data of TFH cells in 3 patients of each group. C, Fluorescence-activated cell sorting analysis of CD191CD2411CD3811 TrB cells of patients with PIDs with PIK3CD and PTEN mutations and control subjects with CVID. D, Representative flow cytometric data of TrB cells in 3 patients of each group.

TSUJITA ET AL 9.e6

9.e7 TSUJITA ET AL

J ALLERGY CLIN IMMUNOL nnn 2016

FIG E6. Classification of patients given a diagnosis of PID according to TREC/KREC levels. A, Patients were classified as follows: TREC1/KREC1: P1-P4, P7, and P9 and TREC2/KREC1: P5, P6, and P8. B, P7 and P9 were classified as TREC1/KREC1, and P8 was classified as TREC2/KREC1. C, P5 was classified as TREC1/KREC1 as a 5-year-old and as TREC2/KREC1 as an 8-year-old.

Macrocephaly, psychomotor retardation Transient pancytopenia, lymphopenia No

Neural complications

2y

Age at evaluation Lymphocyte count (cells/mL) CD31 (% lymphocytes) CD41 (% CD3 cells)

43% to 76%

36% to 75%

40.4%

1,700-6,900

20,500-56,800

71.1%

756

13,700

46.2%

69.6%

1,420

15 y

29,800

0.03 1.71 ND ND ND ND 0.04 <5

PHA (ppm) Lymphocyte subset

10 y

0.6-2.7 5-12.8 0.24-1.35 0.6-4.2 0.1-1.0 0-0.6 0.31-2.02 6.7-1,400

Alive

1.78 25.06 22.41 2.79 0.113 0.007 3.65 1,152

Reference range

IVIG from 10 y

No

Mental retardation, macrocephaly No

ND

Adenoids

Neonatal asphyxia, recurrent LRTI, aspergillosis

2y

Alive

Outcome

Immunologic features Age at evaluation IgM (g/L) (N) IgG (g/L) (N) IgG1 (g/L) (N) IgG2 (g/L) (N) IgG3 (g/L) (N) IgG4 (g/L) (N) IgA (g/L) (N) IgE (IU/mL)

IVIG from 4 to 15 mo and then stopped

Treatment

Other complications

Cytopenia

ND

Mucosal lymphoid hyperplasia

Lymphadenopathy

Neonatal group B Streptococcu infection, Pneumocystis pneumonia, recurrent URTIs SMG, HMG, polyadenopathy

F R15fsX9

M R233X

Infections

B 15

A 3

Kindred Age at diagnosis (y) Sex Mutation

P2

P1

Patients

TABLE E1. Patients’ characteristics

37% to 72%

52% to 78%

1,000-5,300

20,500-56,800

0.63-3.73 12-2,800

1.0-3.8 7.9-17.4

Reference range

No

No

M I5fsX18

10

P3

14 y

ND

0.68 8.84 6.11 2.15 0.87 0.36 1.48 0.93

14 y

48.1%

64.7%

2,030

Alive

Adenomatous goiter, papillary adenocarcinoma of thyroid, glycogenic acanthosis of the esophagus, freckling of the penis Total thyroidectomy

No

Macrocephaly

Multiple gastric polyps

PTEN

37% to 72%

52% to 78%

1,000-5,300

0.69-2.96 7.6-16.8 3.6-20.4 1.6-8.7 0.1-1.2 0-1.6 0.77-3.71 11-2,600

Reference range

C

No

No

M I5fsX18

26

40 y

ND

0.66 10.94 7.15 2.92 0.37 0.58 1.26 2.4

40 y

68.8%

65.8%

1,480

Alive

Total thyroidectomy

Adenomatous goiter, acral keratosis, papillomatous papules on the face

Transient lymphopenia

Intestinal polyposis

P4

39% to 79%

55% to 83%

1,000-2,800

0.67-3.02 7.3-16.3 4.23-10.8 2.65-9.31 0.05-1.21 0.04-1.08 0.83-4.34

Reference range

2.1 10.38 ND 9 y; 0.83 ND ND 9 y; 0.45 9 y; 5.1

1 y, 11 mo

36.6%

68.3%

1,593

8y

17,900

Alive

Rituximab at 8 y, HSCT at 8 y (reject), 9 y

Hematochezia

Cervical lymphadenopathy, adenoidal hypertrophy Follicular lymphoid hyperplasia Macrocephaly, mild mental retardation Thrombocytopenia

Recurrent LRTIs

M E1021K

D 8

P5

39% to 77%

55% to 78%

1,100-5,900

20,500–56,800

9 y; 0.16-1.28 _304 9 y; <

9 y; 1.2-7.0

1 y, 11 mo; 0.57-2.6 1 y, 11 mo; 0.46-12.2

50.1%

58.0%

828

8y

14,600

1.71 6.95 4.16 0.421 0.17 0.03 0.5 <5

3y

IVIG from 3 y, rituximab at 8 y, HSCT at 8 y (reject), 8.5 y (engraft) Alive

Intussusception

Thrombocytopenia Lymphopenia

Follicular lymphoid hyperplasia No

HMG, polyadenopathy

Recurrent LRTIs, otitis media, cellulitis, HSV, HPV

M E1021K

E 9

P6

39% to 77%

55% to 78%

1,100-5,900

20,500–56,800

0.63-2.79 5.3-13.4 2.5-14.1 0.7-4.5 0.1-1.0 0-0.7 0.25-1.74 < _128

M E525A

10

P7

5.59 6.94 4.87 0.12 0.544 0.04 0.29 3

4y

14.5%

93.4%

1,860

10 y

17,444

Alive

None

Hematochezia, growth hormone deficiency

Thrombocytopenia

No

No

SMG, HMG, polyadenopathy

Recurrent LRTIs, otitis media, cellulitis, sepsis, herpes zoster

PIK3CD

37% to 72%

52% to 78%

1,000-5,300

20,500–56,800

0.66-2.88 5.6-13.9 2.6-14.8 0.7-7.4 0.1-1.0 0-0.8 0.31-2.02 < _160

40

P8

40 y

ND

1.78 10.75 8.18 0.98 1.5 <0.004 1.8 ND

40 y

66.5%

55.5%

1,050

Alive

None

No

No

Follicular lymphoid hyperplasia No

Polyadenopathy

Frequent URTIs on childhood

F E525A

F

39% to 79%

55% to 83%

1,000-2,800

0.67-3.02 7.3-16.3 4.23-10.8 2.65-9.31 0.05-1.21 0.04-1.08 0.83-4.34

11 y

ND

1.39 9.58 8.97 <0.66 0.65 <0.004 0.5 ND

12 y

58.5%

68.1%

1,033

Alive

None

No

No

No

ND

Polyadenopathy

No

F E525A

11

P9

(Continued)

37% to 72%

52% to 78%

1,000-5,300

1.0-3.8 7.9-17.4 3.6-20.4 1.6-8.7 0.1-1.2 0-1.6 0.63-3.73

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TSUJITA ET AL 9.e8

PTEN

PIK3CD

CD41 naive (%CD3/CD4 cells) CD81 (% CD3 cells) TFH (%CD3/ CD4 cells) CD19 (% lymphocytes)

70.8%

63% to 91%

40.3%

33% to 66%

58.9%

33% to 66%

27.2%

2.5% to 25%

19.7%

46% to 77%

30.0%

46% to 77%

6.3%

46% to 77%

16.9%

2.5% to 25%

18.3%

46% to 77%

55.8%

22% to 52%

39.8%

13% to 52%

43.5%

13% to 52%

43.7%

14% to 54%

54.9%

28% to 49%

44.6%

28% to 49%

75.9%

13% to 52%

26.6%

14% to 54%

28.2%

13% to 52%

TrB (%CD19 cells) B memory switch (%CD19 cells) CD271IgD2 NK cells (% lymphocytes) NKT cells (% CD3 cells)

0.73% 6.62%

9.64% 10.2% to 18.5%

16.10%

11.2%

5.1% to 10.7%

6.4%

1.5% to 4.1%

3.5%

4% to 23%

0.02%

0.84%

0.32%

10.2% to 15.4%

17.80%

22.7%

3.9% to 7.8%

12.1%

3.9% to 7.8%

7.2%

3.3% to 9.6%

5.2%

3.3% to 9.6%

7.5%

6% to 27%

8.1%

6% to 27%

0.02%

0.04%

10.2% to 15.4%

10.50%

6.41% 11.8%

9.8% 0.01%

15.6% 7.2% to 11.2%

7.9%

1.0% to 3.6% 9.2% to 18.9%

7% to 31%

18.2% 9.8% to 17.7%

23.5%

75.8%

4.5% to 9.2%

3.9%

5.2% to 12.1%

13.7% 0.00%

4% to 26%

23.5%

30.5%

9.8% to 17.7%

2.6%

50.1%

4.5% to 9.2%

46.4%

4.5% to 9.2%

32.4%

1.0% to 3.6%

64.3%

3.7%

5.2% to 12.1%

9.2%

5.2% to 12.1%

57.6%

9.2% to 18.9%

64.9%

3.9% to 7.8% 3.3% to 9.6%

4% to 26%

2.6%

6% to 27%

35.6%

7% to 31%

16.2%

6% to 27%

13.7% 0.00%

0.00%

9.8% to 17.7%

4.7%

13.6%

0.00%

7.2% to 11.2%

7.2%

10.2% to 15.4%

9.e9 TSUJITA ET AL

TABLE E1. (Continued)

0.02%

F, Female; HMG, hepatomegaly; HPV, human papilloma virus; HSCT, hematopoietic stem cell transplantation; HSV, herpes simplex virus; LRTI, lower respiratory tract infection; M, male; MALT, mucosa-associated lymphoid tissue; N, normal value of age; ND, not determined; SMG, splenomegaly; URTI, upper respiratory tract infection.

J ALLERGY CLIN IMMUNOL nnn 2016

PTEN

Patients

P1

Kindred A Age at 4 mo evaluation 2,655 TRECs (copies/ mg DNA) cjKRECs 4,486 sjKRECs 909.8

P2

PIK3CD

P3

Reference Range

B 15 y

5.8 6 2.3 3 10E3

3,430 8.2 6 6.3 3 10E2

P4

P5

P6

40 y

D 8y

E 4y

C Reference range

14 y

Reference range

6,330 8.2 6 6.3 3 10E2

Reference range

428 3.4 6 3.6 3 10E2

Reference range

60.97 2.0 6 1.4 3 10E3

5.8 6 2.4 3 10E4 64,100 1.8 6 0.38 3 10E4 58,100 1.8 6 0.38 3 10E4 39,200 2.1 6 0.44 3 10E4 4,249 1.8 6 0.40 3 10E4 4,760 3.6 6 0.39 3 10E3 7,450 3.6 6 0.39 3 10E3 3,190 1.9 6 0.60 3 10E3 1,015

0

Reference range 3.5 6 2.8 3 10E3

P7

P8

10 y

F 40 y

Reference range

266.3 2.0 6 1.4 3 10E3

1.8 6 0.38 3 10E4 139,997 2.7 6 0.60 3 10E4 121,300 3.6 6 0.39 3 10E3 91,355 9.0 6 1.4 3 10E3 105,900

0

P9 Reference range 3.4 6 3.6 3 10E2

11 y

Reference Range

363.4 2.0 6 1.4 3 10E3

1.8 6 0.38 3 10E4 62,330 2.1 6 0.44 3 10E4 124,500 3.6 6 0.39 3 10E3 33,170 1.9 6 0.60 3 10E3 73,770

1.8 6 0.38 3 10E4 3.6 6 0.39 3 10E3

J ALLERGY CLIN IMMUNOL VOLUME nnn, NUMBER nn

TABLE E2. TREC and KREC levels of the patients

cjKRECs, Coding joint KRECs; sjKRECs, signal joint KRECs.

TSUJITA ET AL 9.e10