# A New Patient with p40phox Deficiency and Chronic Immune Thrombocytopenia

Anna-Lena Neehus, Mathieu Fusaro, Christine Bodemer, Marie Roelens, Adrian Gervais, Jean-Laurent Casanova, Romain Lévy, Jacinta Bustamante

► To cite this version:

Anna-Lena Neehus, Mathieu Fusaro, Christine Bodemer, Marie Roelens, Adrian Gervais, et al. A New Patient with p40phox Deficiency and Chronic Immune Thrombocytopenia. Journal of Clinical Immunology, 2023, 43 (6), pp.1173 - 1177. 10.1007/s10875-023-01498-4. hal-04985753

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# A New Patient with p40<sup>phox</sup> Deficiency and Chronic Immune Thrombocytopenia

Anna-Lena Neehus<sup>1,2</sup> · Mathieu Fusaro<sup>3,4,5</sup> · NCF4 consortium · Romain Lévy<sup>1,2,6</sup> · Jacinta Bustamante<sup>1,2,3,7</sup> ID

Received: 16 February 2023 / Accepted: 19 April 2023 / Published online: 18 May 2023

© The Author(s) 2023

To the Editor,

Chronic granulomatous disease (CGD) is an inborn error of immunity (IEI) caused by deficiencies of any of the five NADPH oxidase subunits (p22<sup>phox</sup> (CYBA), gp91<sup>phox</sup> (CYBB), p47<sup>phox</sup> (NCF1), p67<sup>phox</sup> (NCF2), and p40<sup>phox</sup> (NCF4)) or the chaperone EROS (CYBC1), leading to abolished (classic CGD) or abnormal (variant or partial CGD) reactive oxygen species (ROS) production by the patients' phagocytes [1]. Patients with classic CGD frequently suffer from recurrent, invasive bacterial and fungal infections, whereas patients with autosomal recessive (AR) p40<sup>phox</sup> deficiency have a higher frequency of hyperinflammation and peripheral infections, resembling a milder, variant, atypical form of CGD [1–3].

To date, 26 patients from 14 families with AR p40<sup>phox</sup> deficiency have been described [1, 3]. Most of these patients have inflammatory skin lesions, presenting as lupus-like cutaneous lesions or discoid lupus. About half the patients suffer from inflammatory granulomatous gastrointestinal manifestations, such as inflammatory bowel disease, granulomas, perianal abscesses, and mouth ulcers. Cutaneous infections, mostly due to *Staphylococcus aureus*, were reported in eight of 26 patients, and one patient suffered from disseminated histoplasmosis. The clinical penetrance of p40<sup>phox</sup> deficiency for hyperinflammation or infectious disease is incomplete, at least in childhood, as four of the reported patients remained asymptomatic [1]. Due to the milder clinical phenotype, p40<sup>phox</sup> deficiency is diagnosed later than classic CGD but has a better clinical outcome [1].

We describe here a new case of AR p40<sup>phox</sup> deficiency diagnosed in an 11-year-old girl who had suffered from recurrent mouth ulcers from infancy, chilblain-like purpuric acral lesions, recent photosensitivity, and severe chronic immune thrombocytopenia (ITP). This patient was born to non-consanguineous French parents (Fig. 1a); she was not VZV vaccinated and had experienced an uneventful episode of chicken pox at the age of 4 years. She was vaccinated with the live Bacillus-Calmette Guérin (BCG) vaccine at the age of 5 years and developed local BCG-itis, which resolved spontaneously.

At the age of 8 years, the patient presented thoracic monometameric shingles with painless crusty lesions. Whole blood cell counts revealed marked, isolated thrombocytopenia (28×10<sup>9</sup>/L), with other blood cell counts within the normal range for the age of the patient (leukocytes: 8800/mm<sup>3</sup>; neutrophils (PMNs): 3000/mm<sup>3</sup>; lymphocytes: 4500/mm<sup>3</sup>; monocytes: 700/mm<sup>3</sup>). The hemoglobin concentration was also normal (11.9 g/dL). Immunophenotyping revealed the proportions of total T, B, and NK cells to be normal, with abnormally high proportions of naïve CD4<sup>+</sup> T cells and effector memory CD8<sup>+</sup> T cells and low proportions of central memory and TEMRA CD8<sup>+</sup> T cells (Table S1).

Romain Lévy and Jacinta Bustamante equally contributed to this study.

<figure><img src="image_2.png" /></figure>

Jacinta Bustamante
jacinta.bustamante@inserm.fr

<footnote n="1">¹ Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France, EU</footnote>

<footnote n="2">² Paris Cité University, Imagine Institute, Paris, France, EU</footnote>

<footnote n="3">³ Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, Paris, France, EU</footnote>

<footnote n="4">⁴ Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), INSERM U1291, CNRS U5051, University Toulouse III, Toulouse, France, EU</footnote>

<footnote n="5">5 Laboratory of Immunology, CHU Purpan Toulouse, INSERM U1291, CNRS U5051, University Toulouse III, Toulouse, France, EU</footnote>

<footnote n="6">⁶ Pediatric Immunology-Hematology and Rheumatology Unit, Necker Hospital for Sick Children, AP-HP Paris, France, EU</footnote>

<footnote n="7">⁷ St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA</footnote>
<figure><img src="image_3.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>h</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>i</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>k</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>m</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>j</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>l</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>n</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>o</figcaption></figure>

<figure><img src="image_17.png" /></figure>
►Fig. 1 Identification of a new patient with p40phox deficiency. a Pedi-
gree of the family showing familial segregation of the NCF4 alleles.
b Chilblain-like lesions on the patient's extremities (left) and mouth
ulcers (right). c Schematic representation of the patient's clinical
history. d Alamut viewer presentation of the NCF4 gene containing
the variants for patient e quantification of DHR means fluorescence
intensity (MFI) of control (Ctrl) and patient (P) neutrophils (PMN)
and monocytes (Mono) after stimulation with PMA or E. coli, rela-
tive to unstimulated control. f Western blot for p40phox in control
(Ctrl) and patient (P) neutrophils (PMN). Extracellular H2O2 release
and p40phox expression by g, h M-CSF-IL-4-derived macrophages
(M-CSF/IL-4 MΦ); i, j GM-CSF-derived macrophages (GM-CSF
MΦ); and k, l IL-34-derived macrophages (IL-34 MΦ) from a
healthy control (Ctrl) and the patient (P); technical duplicates ± SD.
Extracellular H2O2 release (m) and p40phox expression (n) by mono-
cyte-derived dendritic cells (MDDC) from a healthy control (Ctrl)
and the patient (P); technical triplicates ± SD. o Luciferase-based
neutralization assay to detect auto-Abs neutralizing 10 ng/mL or
100 pg/mL IFN-α2 using plasma from healthy controls (Ctrl), the
p40phox-deficient patient (P), and an individual known to have neutral-
izing IFN-α2 auto-Abs (Ctrl+) at a 1:10 dilution. A relative luciferase
activity (RLA) <15% of the value for the mock treatment was consid-
ered to correspond to neutralizing activity (dashed line)

Extensive B-cell immunophenotyping revealed normal
proportions of naïve and memory B cells, with no excess
of CD21lowCD38low B cells. Immunoglobulin levels were
within the normal range for IgG (12.12 g/L), IgA (1.38 g/L),
and IgM (1.52 g/L). Both CRP (2.2 mg/L) and fibrinogen
(2.5 g/L) concentrations were normal. The patient tested
negative for anti-extractable nuclear antigen, anti-DNA,
and anti-Smith autoantibodies. A slightly positive result was
obtained for anti-platelet antibodies in the monoclonal anti-
body-specific immobilization of platelet antigens (MAIPA)
assay. A blood smear revealed no morphological abnormali-
ties in the platelets and no schistocytes. The patient also
presented recurrent severe mouth ulcers but was otherwise
well (Fig. 1b, c). No mucosal bleeding, hematomas, hepato-
splenomegaly, or adenopathy was observed.

At the age of 9 years, the patient developed a local thigh
abscess due to methicillin-resistant S. aureus, which was
treated surgically and did not recur. Two months later, she
developed lupus-like vasculitis with chilblain-like lesions
on her extremities (Fig. 1b, c). The lesions improved rapidly
on hydroxychloroquine and betamethasone. Hydroxychloro-
quine was discontinued after 6 months, and the acral lesions
had not recurred at the last follow-up. However, the patient's
thrombocytopenia worsened over time (<10×10⁹/L), with
a transient partial (55×10⁹/L) response to intravenous
immunoglobulin, necessitating second-line weekly subcu-
taneous romiplostim injections (5 µg/kg/week), which led
to a durable complete response. A bone marrow aspiration,
performed at the age of 9, showed a polymorphic-rich bone
marrow containing megakaryocytes and without cytological
abnormalities or signs of hemophagocytosis. Additionally,
liver testing for aspartate transaminases (AST) (38 U/L) and
alanine transaminases (ALT) (20 U/L) gave normal results,

and splenomegaly was ruled out by performing a CT scan
at the age of 9. At the last follow-up, the patient was doing
well, with no complaints other than a few recurrent mouth
ulcers (about two small lesions per month) and mild photo-
sensitivity, and is currently on low-dose romiplostim (3 µg/
kg/week), which has made it possible to maintain platelet
count at 105×10⁹/L. Both her parents and her older brother
are healthy.

An IEI was suspected, and the patient was genetically investigated by whole-exome sequencing (WES). Two heterozygous variants were found in intron 6 and exon 7 of *NCF4* in her WES data: c.577del and c.529-2A > T (NM_013416.3), respectively (Fig. 1d). No non-synonymous or copy number variants of any other genes underlying known IEI and compatible with the patient's clinical phenotype were identified. Familial segregation confirmed an AR mode of inheritance, with the c.577del variant inherited from the patient's father and the c.529-2A > T from her mother (Fig. 1a). Both variants were predicted to be deleterious, with the first creating a frameshift and a premature stop codon (p.A193Lfs*4) and the second disrupting the splicing acceptor site at the junction of intron 6 and exon 7.

An investigation of the patient's circulating phagocytes
revealed abnormally low levels of ROS production by PMNs
and monocytes in response to phorbol 12-myristate 13-ace-
tate (PMA) or opsonized *Escherichia coli* (Fig. 1e). These
findings were consistent with previous reports showing that
AR p40phox deficiency leads to the impairment, but not total
abolition, of ROS production in PMNs and monocytes [1–3].
Western blotting of whole-cell lysate of the patient's PMNs
showed an absence of detectable p40phox protein, whereas
it was readily detectable in healthy control (Fig. 1f). Thus,
both *NCF4* variants carried by the patient are deleterious and
result in a complete absence of the protein.

We then evaluated the impact of p40phox deficiency in
monocyte-derived phagocytes. Using isolated CD14+ mono-
cytes from the patient and healthy control, we generated
three different types of macrophages by priming the cells
with GM-CSF (GM-CSF MΦ) to induce pro-inflammatory
macrophages, or IL-34 (IL-34 MΦ) or M-CSF in combi-
nation with IL-4 (M-CSF/IL-4 MΦ), to favor an M2-like
polarization of the macrophages. Consistent with the find-
ings of a previous study, M-CSF/IL-4 MΦ generated from
the patient's monocytes displayed normal ROS produc-
tion (Fig. 1g) [1]. The absence of p40phox protein in the
patient's cells was further confirmed by western blotting
(Fig. 1h). Interestingly, ROS production was not affected in
p40phox-deficient GM-CSF MΦ relative to healthy controls
(Fig. 1i, j). Similarly, IL-34 MΦ lacking p40phox protein dis-
played normal ROS production after stimulation with PMA
in combination with IFN-γ priming (Fig. 1k, l).

We also investigated extracellular ROS production by
monocyte-derived dendritic cells (MDDCs) from the patient
and found that it was similar to that of control cells after stimulation with PMA (Fig. 1m). The priming of MDDCs with lipopolysaccharide (LPS) for 24 h led to similar levels of ROS production in both patient and control MDDCs, even in the absence of p40<sup>phox</sup> protein expression (Fig. 1n). These results suggest that p40<sup>phox</sup> deficiency does not affect the production of H<sub>2</sub>O<sub>2</sub> in various subsets of monocyte-derived cells. We did not explore the role of p40<sup>phox</sup> in tissue-resident macrophages or dendritic cells, which could be more relevant for host defense against intracellular pathogens, including mycobacteria [4–6], due to a lack of patient material available from which these cells could be isolated.

The patient presented with herpes zoster at the age of 8 years, which has not been reported previously in patients with p40<sup>phox</sup> deficiency and very rarely in patients with other genetic etiologies conferring CGD. We tested the patient's plasma for the presence of auto-antibodies (auto-Abs) neutralizing type I IFNs, which were previously shown to underlie viral disease, including herpes zoster [7]. The patient's plasma contained neutralizing auto-Abs against IFN-α2, neutralizing a dose of 100 pg/mL when used at a 1:10 dilution, which thus could underlie the viral disease observed in this patient (Fig. 1o).

Cutaneous infections and skin inflammation have been observed in more than a third of the reported p40<sup>phox</sup>-deficient patients, but this patient is the first to present with chronic ITP [1–3]. ITP is a frequent complication of systemic lupus erythematosus and has been observed only rarely in patients with classic CGD or female carriers of heterozygous mutations of *CYBB* [8]. Both these groups are at risk of developing autoimmune manifestations, probably due to their abnormal ROS production in B cells [9]. Patients with classic CGD have been reported to have a low frequency of memory B cells, whereas this patient with atypical CGD displayed no changes in B-cell phenotype during follow-up. Additional studies on B cells from p40<sup>phox</sup>-deficient patients and other forms of CGD would help to elucidate the mechanism underlying autoimmunity in these patients.

The clinical penetrance of lupus-like symptoms is incomplete in p40<sup>phox</sup>-deficient patients, and additional follow-up studies are required to characterize the clinical spectrum of this disease in more detail, together with the underlying molecular mechanisms. P40<sup>phox</sup> deficiency should be considered in patients with ITP with no clear differential diagnosis to ensure its early detection and appropriate treatment.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s10875-023-01498-4.

**Acknowledgements** We thank the patients and their families for participating in this study. We thank all members of the Laboratory of Human Genetics of Infectious Diseases for helpful input and discussions. We thank Lazaro Lorenzo-Diaz, Yelena Nemirovskaya, Dana Liu, and Maya Chrabieh for administrative support.

**NCF4 consortium**

Christine Bodemer<sup>2,8</sup>

Marie Roelens<sup>2,3</sup>

Adrian Gervais<sup>1,2</sup>

Jean-Laurent Casanova<sup>1,2,7,9,10</sup>

<sup>8</sup>Department of Dermatology, Necker Hospital for Sick Children, AP-HP, Paris, France, EU

<sup>9</sup>Department of Pediatrics, Necker Hospital for Sick Children, AP-HP, Paris, France, EU

<sup>10</sup>Howard Hughes Medical Institute, New York, NY, USA

**Author Contribution** A.-L.N., R.L., and J.B. wrote and edited the manuscript, along with the NCF4 consortium. A.-L.N. performed experiments. A.G. evaluated the presence and neutralization of auto-Abs. M.F. and J.B. performed a genetic investigation of the patient. All the authors have read and approved the final manuscript.

**Funding** This study was supported in part by a grant from the St. Giles Foundation, the Rockefeller University, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris Cité University, the National Center for Research Resources, the National Center for Advancing Sciences of the National Institutes of Health (NIH; UL1TR001866), the National Institute of Allergy and Infectious Diseases, NIH (R01AI095983), the French National Research Agency (ANR) under the "Investments for the Future" program (ANR-10IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID) and the ANR project MAFMACRO (ANR-22-CE92-0008), the French Foundation for Medical Research (EQU201903007798), and the Square Foundation. A-L.N. is supported by the Bettencourt-Schueller Foundation, the International PhD program of the Imagine Institute, and the fin de thèse program of the Fondation pour la Recherche Médicale (FDT202204015102). M.F. is supported by the Fondation pour la Recherche Médicale (FDM202006011216).

**Data Availability** All data and materials can be obtained by contacting the corresponding author.

**Declarations**

**Ethics Approval** Written informed consent was obtained in accordance with local regulations, with approval from the institutional review board (IRB). The experiments were performed with the approval of the IRB Ile de France II in France (no. ID-RCB: 2010-A00850-39).

**Consent to Participate** Written informed consent for participation was obtained from the patient's parents.

**Consent for Publication** Consent for publication was obtained from the patient's parents. All the authors approved the final version of the manuscript.

**Competing Interests** The authors declare no competing interests.

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