# Endophilin A2 Deficiency Impairs Antibody Production in Humans

Cybel Mehawej, Eliane Chouery, Roula Farah, Alia Khalil, Setrida El Hachem, Sandra Corbani, Valérie Delague, Issam Mansour, Tarek Najemdeen, Rima Korban, et al.

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Cybel Mehawej, Eliane Chouery, Roula Farah, Alia Khalil, Setrida El Hachem, et al.

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# Endophilin A2 Deficiency Impairs Antibody Production in Humans

Cybel Mehawej<fnref n="1" /> · Eliane Chouery<fnref n="1" /> · Roula Farah<fnref n="2" /> · Alia Khalil<fnref n="3" /> · Setrida El Hachem<fnref n="1" /> · Sandra Corbani<fnref n="1" /> · Valerie Delague<fnref n="4" /> · Issam Mansour<fnref n="5" />,<fnref n="6" /> · Tarek Najemdeen<fnref n="6" /> · Rima Korban<fnref n="1" /> · Wissam H. Faour<fnref n="3" /> · Gerard Lefranc<fnref n="7" /> · Andre Megarbane<fnref n="1" />,<fnref n="8" />

Received: 13 August 2024 / Accepted: 19 October 2024

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024

## Abstract

Endophilin A2, the sole endophilin A family member expressed in hematopoietic cells, regulates various aspects of membrane dynamics, including autophagy and endocytosis. Recent studies in rodents highlight the essential role of endophilin A2 in modulating immune responses. Here we report a homozygous frameshift variant in the *SH3GL1* gene (NM_003025.3:c.427delC; p.Leu143Serfs*9), detected by whole exome sequencing in a 14-year-old boy with predominantly antibody deficiency. The patient who is issued from a consanguineous Lebanese family, presents since the age of 18 months with recurrent respiratory tract infections, low peripheral B cell counts and pan-hypogammaglobulinemia, with no history of opportunistic infections. This defect is associated with decrease in switched memory B cells development, impaired in-vitro B cell proliferation and diminished in-vitro IgG production. The detected variant in *SH3GL1* segregates with the disease in the family. It significantly decreases the expression of the protein in the patient's peripheral blood compared to healthy controls, thus confirming its pathogenicity. Interestingly, endophilin A2-deficient *Sh3gl1*⁻/⁻ mice have been reported to present defects in germinal center B cell responses and in the production of high-affinity IgG. Our data suggests that endophilin A2 deficiency impairs antibody production in humans. Reporting further cases with mutations in *SH3GL1* is needed to better characterize the inborn error of immunity linked to this gene.

**Keywords** Predominantly antibody deficiency · Endophilin A2 · SH3GL1 · Exome sequencing

## Introduction

Predominantly or Primary antibody deficiencies (PAD) represent the most common form of inborn errors of immunity (IEI) globally [1]. This heterogenous group of diseases is characterized by decreased or abolished production of one

or more of the different immunoglobulins classes, in the presence or absence of B cells [2]. PAD include: defects in early B cell development, class switch recombination defects (hyper-IgM syndromes), and common variable immunodeficiency phenotypes (CVID); in addition to isotype, light chains or functional deficiencies [1, 3].

<footnote>☒ Cybel Mehawej
cybel.mehawej@lau.edu.lb</footnote>

<footnote>☒ Andre Megarbane
andre.megarbane@lau.edu.lb</footnote>

<footnote n="1">¹ Department of Human Genetics, Gilbert and Rose-Marie Chagoury School of Medicine, Lebanese American University, Byblos, Lebanon</footnote>

<footnote n="2">² Department of Pediatrics, Gilbert and Rose-Marie Chagoury School of Medicine, Lebanese American University, Byblos, Lebanon</footnote>

<footnote n="3">³ Gilbert and Rose-Marie Chagoury School of Medicine, Lebanese American University, Byblos, Lebanon</footnote>

<footnote n="4">⁴ Aix Marseille Univ, INSERM, MMG, Marseille U 1251, France</footnote>

<footnote n="5">⁵ Molecular Biology Laboratory, Faculty of Health Sciences, American University of Science and Technology, Beirut, Lebanon</footnote>

<footnote n="6">⁶ Inovie Laboratory, Fayadieh, Lebanon</footnote>

<footnote n="7">⁷ The International ImMunoGeneTics Information System®, Laboratoire d'ImmunoGénétique Moléculaire, Institut de Génétique Humaine, UMR 9002 CNRS, Université de Montpellier, Montpellier cedex 5, France</footnote>

<footnote n="8">⁸ Institut Jérôme Lejeune, Paris, France</footnote>
The clinical spectrum of patients with PAD ranges from severe disease in patients with agammaglobulinemia to mild disease in cases with selective immunoglobulin deficiencies [2, 4]. The hallmark of PAD is increased susceptibility to infections with encapsulated bacteria, mainly affecting the respiratory tract and leading, if untreated, to severe bronchiectasis and sinusitis. Less frequently, some cases with PAD suffer from intestinal infections and bacterial cutaneous infections [4–6]. Additionally, enteropathy, autoimmunity, autoinflammation, lymphoproliferation and allergic diseases can manifest in patients with PAD [4, 6]. Management of these cases mainly relies on lifelong immunoglobulin replacement, combined, as needed, with antimicrobial agents. Close monitoring of patients is crucial to allow early management of associated complications [2].

Advances in the immunogenetics field enabled the identification of more than 40 PAD genes [1], and the elucidation of their molecular pathways. These can be B cell-intrinsic (defects in B cell development, migration, survival, activation or class-switching) or B cell-extrinsic (impairment in innate immune cells or T cells) defects. To date, the genetic basis in many patients with PAD remains uncharacterized, particularly in patients with CVID [6].

In Lebanon, as in the Middle East and North Africa (MENA) region, the high consanguinity rate favors the occurrence of rare autosomal recessive diseases, enabling the identification of novel genetic causes of IEI [5]. Here we report a homozygous frameshift variant in *SH3GL1* encoding endophilin A2, in a 14-year-old boy with decreased peripheral B cell number and panhypogammaglobulinemia. No germline variants in this gene have been previously reported in humans. *SH3GL1* encodes endophilin A2, a 42KDa protein, comprising 368 amino acids [7]. It is the sole endophilin-A family member expressed in hematopoietic cells [8]. Endophilins are Bin/Amphiphysin/Rvs (BAR) and SH3 domain-containing proteins [9], that modulate the dynamics of intracellular compartments, participating in processes such as membrane curvature, vesicle trafficking, and cell signaling [10]. Endophilin A2 plays a crucial role in the regulation of a clathrin-independent endocytic pathway known as fast endophilin-mediated endocytosis (FEME) [11]. While previous studies mainly focused on the involvement of endophilin A2 in central nervous system processes [12, 13], and in cancer [14, 15], recent findings in rodents have underscored the essential role of endophilin A2 in modulating immune responses [8, 16]. Endophilin A2-deficient *Sh3gl1*⁻/⁻ mice show defects in germinal center B cell responses and in high-affinity IgG production [8], which is consistent with the phenotype of the patient herein reported.

# Methods

## Study Subjects

The proband is a 14-year-old male child of first-cousin consanguineous Lebanese parents. He underwent a work-up including clinical evaluation, molecular analyses, and blood tests (complete blood count, serum immunoglobulin testing and blood immunophenotyping by flow cytometry). Written informed consent from the patient's parents and assent from the patient and his unaffected sibling were obtained. Regarding enrollment of healthy controls, consent forms were obtained from healthy adult controls and from the parents of the healthy age-matched controls. Assent forms were also obtained from healthy age-matched controls to enable their contribution to the study.

## Genetic Studies

### a. DNA extraction.

EDTA blood samples from all members of the family were collected for genetic studies. Genomic DNA was isolated from peripheral blood by standard salt-precipitation methods.

### b. Whole Exome Sequencing (WES):

WES was carried-out in the patient. Exome was captured and enriched using the solution Agilent SureSelect Human All Exon kit version 5.0 and samples were then multiplexed and subjected to sequencing on an Illumina HiSeq 2500 PE100-125. Reads files (FASTQ) were generated from the sequencing platform via the manufacturer's proprietary software. Reads were aligned to the hg19/b37 reference genome using the Burrows-Wheeler Aligner (BWA) package version 0.7.11 [17]. Variant calling was subsequently performed using the Genome Analysis Tool Kit (GATK) version 3.3 [18]. Variants were called using high stringency settings and annotated with VarAFT software 1.61 [19] containing information from dbSNP147 and the Genome Aggregation database (gnomAD, http://gnomad.broadinsti.tute.org). Only nonsynonymous coding and splicing variants found in the patient were considered. Variant filtering was performed according to the mode of transmission of the disease in the family, the frequency of the variant in the gnomAD database (<0.01% and <50 heterozygous carriers or <5 homo-/hemizygous carriers), and in our in-house database (<1 homozygous occurrence).

### c. Sanger Sequencing:
The selected variant was studied in the proband, his parents and his unaffected sister by Sanger sequencing. Genomic and cDNA sequences of *SH3GL1* (NM_003025.3) were obtained from UCSC Genomic Browser. Primers used for PCR amplification were designed using Primer3 software (http://frodo.wi.mit.edu) to amplify the region surrounding the mutation detected by exome sequencing. PCR products were purified by exonuclease I/Shrimp Alakaline Phosphatase treatment (ExoSAP-IT; Fisher Scientific SAS, Illkirch, France) according to the manufacturer's instructions and both strands were sequenced using the Big Dye® Termi-nator v1.1 Cycle Sequencing Kit (Applied Biosystems). Sequence reactions were purified on Sephadex G50 (Amersham Pharmacia Biotech, Foster City, CA) and capillary electrophoresis was performed on Genetic Analyser 3100 (Applied Biosystems). Electropherograms were analyzed on the Sequence Analysis Software version 5.2 (Applied Biosystems) and aligned with the wild-type *SH3GL1* gene sequence using ChromasPro version 1.22 (Technelysium, Queensland, Australia).

## Cell Culture and Cell Stimulations

Human PBMCs were isolated from heparinized blood obtained from the patient, his parents and healthy age-matched controls using Ficoll-Hypaque (GE Healthcare).

For B cell stimulations, human PBMCs were stimulated with anti-CD40 (GTX14148, GeneTex) at 10 µg/ml and IL-21(200–21, PeproTech) at 50 ng/ml. The duration of the stimulation was 3 days for the proliferation assay, while it was 7 days for the in-vitro IgG production. In other words, each experimental condition, involving both patient and control cells, was performed in duplicates: one set was analyzed via flow cytometry on day 3 for proliferation assessment, while the other set was maintained for 7 days, after which supernatants were collected to assess IgG production.

For T cell stimulations (for the proliferation assay), human PBMCs were stimulated three days with purified anti-human CD3 antibody (#300401, Biolegend) used at 100 ng/ml and purified anti-CD28 human antibody (#302901, Biolegend) at 2 µg/ml; or with phytohemagglutinin (PHA-P, #inh-phap, Invivogen) at 1 µg/ml; or with Phorbol Myristate Acetate (PMA, #tlrl-pma, Invivogen) at 20 ng/ml and ionomycin (#FL162671, Biosynth) at 0.5 µM.

## Enzyme Linked Immunosorbent Assay (ELISA)

In-vitro production of IgG was assessed by ELISA, following 7 days of stimulation of PBMCs with anti-CD40 (GTX14148, GeneTex) at 10 µg/ml and IL-21(200–21, PeproTech) at 50 ng/ml. Supernatants were collected at day 7 and used for IgG ELISA. ELISA was performed using

Human IgG (Immunoglobulin G) ELISA Kit (#E-EL-H0169, Elabscience) as per manufacturer instructions.

## Proliferation Assay/CFSE Staining

In-vitro proliferation assays were performed using eBioscince CellTrace Carboxyfluorescein succinimidyl ester (CFSE) proliferation dye (65-0850-84, Invitrogen). Briefly, PBMCs were stained with the CFSE dye at a final concentration of 5 µM in PBS1X solution containing 0.1% BSA. Cells were incubated at 37 °C for 7 min to allow staining. Then, to halt the staining process, cells were washed with RPMI. Cells were then plated in round bottom culture plates in media containing when indicated appropriate stimuli and incubated at 37 °C. Cell proliferation was assessed by flow cytometry on day 3.

## Immunoblot Analysis

Total protein was extracted from PBMCs from the patient, his parents and two healthy controls. Cell lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes. Non-specific binding was blocked by incubating membranes 1 h at room temperature in 5% non-fat dry milk in Tris Buffered Saline-Tween. Blots were then probed with primary antibodies, overnight at 4 Celsius degrees. Primary antibodies used are SH3GL1 antibody- N-terminal region (1:500; #ARP56547; Aviva Systems biology) targeting the peptide sequence (71–120; in the canonical isoform; region located upstream of the variant p.Leu143Serfs*9); and beta-Actin antibody (13E5) (1:1000; #4970, Cell signaling). Secondary antibody used is peroxidase affinipure goat anti-rabbit IgG (1:10000; 111-035-003, Jackson immunoresearch). Antibody binding was detected by western lightning plus-ECL, enhanced chemiluminescence substrate (PerkinElmer). Specific protein expression was first normalized to endogenous beta-Actin expression, in each individual. The relative expression of the normalized protein was then assessed in each individual, in comparison to the average expression in the healthy controls.

## Results

### Identification of a Homozygous Frameshift Variant in the SH3GL1 Gene in a Patient with Predominantly Antibody Deficiency

The patient is a 14-year-old boy born to first-cousin Lebanese parents (Fig. 1A). He was born at term at 39 weeks of gestation after an unremarkable pregnancy. His birth weight was 3030 g, length was 49 cm, and head circumference was
<figure><img src="image_2.png" /><figcaption>**Fig. 1** Homozygous *SH3GL1* variant in a patient with a primary antibody deficiency. A: Pedigree of the family included in this study. B: Sanger sequencing confirming the segregation of the *SH3GL1* variant (NM_003025.3:c.427delC; p.Leu143Serfs*9) with the disease in the family. The red arrow pinpoints the c.427delC variant in *SH3GL1* that is heterozygous in parents (III-1 and III-2) and in the unaffected sibling (IV-1) and is homozygous in the proband (IV-2). Y: heterozygous peak. C: Immunoblots analysis of SH3GL1 showing the significant</figcaption></figure>

<figure><img src="image_3.png" /></figure>

decrease in protein expression in PBMCs from the patient, compared to his parents and two healthy controls. Quantitation of endophilin A2 (SH3GL1) protein expression in PBMCs of patient, his parents and healthy controls, normalized for β-actin expression and expressed as fold change compared to the average of the value of healthy control PBMCs. Results represent means ± S.E.M. \*\*p < 0.01; \*\*\*\*, p < 0.0001, by one-way ANOVA

33 cm (all measurements within normal limits). Developmental milestones were within normal ranges. At 10 months of age, he was admitted to the hospital for meningitis of unknown cause, for which he received intravenous antibiotics. At 18 months old, he started experiencing recurrent respiratory tract infections and bronchiectasis, leading to three or more hospitalizations per year. He did not exhibit any opportunistic infection. Blood, stool and urine cultures were reported negative. At 6 and a half years of age, flow cytometry revealed low peripheral B cell number (according to the referring physician who has been following the patient since childhood), and serum immunoglobulins testing showed panhypogammaglobulinemia: IgG levels were 146 mg/dL (normal range: 700-1600 mg/dL), IgM were 38 mg/dL (normal range: 55-264 mg/dL), and IgA were 14 mg/dL (normal range: 28-256 mg/dL). A diagnosis of PAD was established and the patient was put on a regimen of intravenous immunoglobulin (IVIG; at 0.5 g/kg/dose) treatment, every four weeks. This treatment was highly effective, leading to a significant reduction in infection rates and no hospitalizations, except for one instance at age 12 when he tested positive for influenza A. Bone marrow aspirate

performed at the age of 10 years following the detection of low white blood count revealed normal results with no blasts increase. At 12 years old, he developed thrombocytopenia with platelet levels reaching 64,000 cells/µL. A repeat bone marrow aspirate smear was normal.

When the patient presented to us at 14 years old, he had decreased white blood cell count (3.41×10³/µL; normal range: 5-13×10³/µL), slightly low lymphocytes count (0.89×10³/µL; normal range: 0.9-5.2×10³/µL) and thrombocytopenia (80×10³/µL; normal range: 150-400×10³/µL). Lymphocyte phenotyping revealed significant reduction in the number of circulating B cells, with decreased switched memory B cells and increased naïve B cells counts, compared to normal ranges (Table 1). The patient also exhibited mild T cell lymphopenia, particularly modestly decreased CD8 T cell counts. Immunophenotyping showed a consistent increase in effector memory CD4⁺ T cells, decrease in central memory CD4⁺ T cells; decrease in central memory CD8⁺ T cells and increase in CD8⁺ T effector Memory RA⁺ Cells (TEMRAs) (Table 1). Serum immunoglobulin levels performed three weeks after administration of his immunoglobulin replacement therapy
<table><caption>**Table 1** Immune phenotyping of the patient's lymphocytes. Bolded values are outside the normal range</caption><thead><tr><th>Age at the time of testing (years)</th><th>P</th><th>P</th></tr></thead><tbody><tr><td></td><td>13.5</td><td>14</td></tr><tr><td><strong>Hemogram (normal range)</strong></td><td></td><td></td></tr><tr><td>Hemoglobin, g/dL</td><td>12.6 (13–16)</td><td>11.9 (13–16)</td></tr><tr><td>WBCs, 10<sup>3</sup> cells/µL</td><td><strong>3.41</strong> (5–13)</td><td><strong>4.58</strong> (5–13)</td></tr><tr><td>Neutrophils, 10<sup>3</sup> cells/µL</td><td>2.18 (1.9–8)</td><td>2.89 (1.9–8)</td></tr><tr><td>Lymphocytes, 10<sup>3</sup> cells/µL</td><td><strong>0.89</strong> (0.9–5.2)</td><td>1.08 (0.9–5.2)</td></tr><tr><td>Monocytes, 10<sup>3</sup> cells/µL</td><td>0.27 (0.16–1)</td><td>0.39 (0.16–1)</td></tr><tr><td>Platelets, 10<sup>3</sup> cells/µL</td><td><strong>80</strong> (150–400)</td><td><strong>70</strong> (150–400)</td></tr><tr><td><strong>Lymphocyte subsets</strong></td><td></td><td></td></tr><tr><td>CD3<sup>+</sup>, 10<sup>3</sup> cells/µL</td><td><strong>649</strong> (850–3200)</td><td><strong>588</strong> (850–3200)</td></tr><tr><td>CD3<sup>+</sup>CD4<sup>+</sup>, 10<sup>3</sup> cells/µL</td><td>413 (400–2100)</td><td>420 (400–2100)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> Naïve, % CD4<sup>+</sup></td><td>54 (39.50–66.26)</td><td><strong>38</strong> (39.50–66.26)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> Effector memory, % CD4<sup>+</sup></td><td><strong>38</strong> (4.68–15.70)</td><td><strong>50</strong> (4.68–15.70)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> Central memory, % CD4<sup>+</sup></td><td><strong>7</strong> (25.34–49.90)</td><td><strong>5</strong> (25.34–49.90)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> Effector, % CD4<sup>+</sup></td><td>1 (0.00–1.54)</td><td><strong>8</strong> (0.00–1.54)</td></tr><tr><td>CD3<sup>+</sup>CD8<sup>+</sup>, 10<sup>3</sup> cells/µL</td><td><strong>257</strong> (300–1300)</td><td><strong>161</strong> (300–1300)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> Naïve, % CD8<sup>+</sup></td><td>53 (35.34–72.32)</td><td>41 (35.34–72.32)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> Effector memory, % CD8<sup>+</sup></td><td>2 (2.38–15.84)</td><td>4 (2.38–15.84)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> Central memory, % CD8<sup>+</sup></td><td><strong>1</strong> (10.96–31.00)</td><td><strong>0</strong> (10.96–31.00)</td></tr><tr><td>CD45RA<sup>+</sup>CCR7<sup>+</sup> TEMRA, % CD8<sup>+</sup></td><td><strong>44</strong> (5.08–31.24)</td><td><strong>55</strong> (5.08–31.24)</td></tr><tr><td>CD19<sup>+</sup>, 10<sup>3</sup> cells/µL</td><td><strong>83</strong> (160–700)</td><td><strong>46</strong> (160–700)</td></tr><tr><td>CD27<sup>−</sup> IgD<sup>+</sup> Naïve B cells, % CD19<sup>+</sup></td><td><strong>92</strong> (61.6–87.4)</td><td><strong>89</strong> (61.6–87.4)</td></tr><tr><td>CD27<sup>+</sup> IgD<sup>+</sup> Unswitched Memory B cells, % CD19<sup>+</sup></td><td>6 (2.6–13.4)</td><td><strong>2</strong> (2.6–13.4)</td></tr><tr><td>CD27<sup>+</sup> IgD<sup>−</sup> Switched Memory B cells, % CD19<sup>+</sup></td><td><strong>1</strong> (4.0–21.2)</td><td><strong>2</strong> (4.0–21.2)</td></tr><tr><td>CD27<sup>−</sup> IgD<sup>−</sup> Late Memory B cells, % CD19<sup>+</sup></td><td><strong>1</strong> (1.4–13.0)</td><td>7 (1.4–13.0)</td></tr><tr><td>CD3<sup>−</sup>CD56<sup>+</sup>CD16<sup>+</sup>, 10<sup>3</sup> cells/µL</td><td>0.094 (0.092–1.2)</td><td><strong>0.08</strong> (0.092–1.2)</td></tr><tr><td><strong>Immunoglobulins</strong></td><td></td><td></td></tr><tr><td>IgG, g/L</td><td>13.5 (5.53–13.07)</td><td><strong>4.97</strong> (5.53–13.07)</td></tr><tr><td>IgM, g/L</td><td><strong>&lt;0.18</strong> (0.490–2.612)</td><td><strong>0.29</strong> (0.490–2.612)</td></tr><tr><td>IgA, g/L</td><td><strong>&lt;0.26</strong> (0.765–2.192)</td><td><strong>0.28</strong> (0.765–2.192)</td></tr></tbody></table>

showed decreased IgA and IgM levels but normal IgG levels (Table 1). Specific antibody response to vaccines was not assessed. ANA testing was negative.

Exome sequencing (ES) in the patient revealed a homozygous variant in *SH3GL1* (NM_003025.3:c.427delC; p.Leu143Serfs*9). This frameshift variant has a CADD score of 25.1 and is predicted to be pathogenic as per the ACMG guidelines (PVS1, PM2). It was only detected once at a heterozygous state in gnomAD_V4 (1/1613514 alleles), and is absent in our in-house database including 980 individuals. Sanger sequencing confirmed the segregation of the p.Leu143Serfs*9 variant with the disease in the family, as both parents and the unaffected sister are heterozygous carriers, while the patient is homozygous for the detected variant (Fig. 1A and B). On the other hand, all IEI genes were fully covered at a minimum of 20X, with no “non benign” variants in the patient, except for a heterozygous pathogenic variant in *MEFV* (NM_000243.2:c.2177T > C; p.Val726Ala), that could not be correlated with the patient’s manifestations. Additional rare homozygous variants identified by ES are listed in Table 2. These were excluded based on the corresponding gene function, its expression pattern,

and/or previous disease associations. For instance, a frameshift variant in *ADCK1*, a gene important for mitochondrial function, was initially considered because of the importance of mitochondria in B and T cell homeostasis and function. The variant is detected at a heterozygous state in two individuals of our in-house database that comprises 980 individuals, rendering its allele frequency equal to approximately 0.001. However the gene belongs to a protein family that has been linked to mitochondriopathies mainly affecting the brain, muscles and kidneys [20–22], systems that are not affected in our patient.

## The p.Leu143Serfs*9 Variant in SH3GL1 Abolishes the Expression of the Protein in the Patient PBMCs

*SH3GL1* encodes endophilin A2, a 42kDa protein, comprising 368 amino acids. To assess the impact of the frameshift variant in *SH3GL1* on the expression of the protein, immunoblotting for endophilin A2 was performed on PBMC lysates from the patient and two healthy controls, using a N-terminal SH3GL1-specific antibody targeting the peptide sequence 71–120 of the canonical isoform of the protein
<table><caption>**Table 2** List of rare homozygous variants detected in the patient by ES</caption><thead><tr><th>gene</th><th>Variant at the cDNA level</th><th>Variant at the protein level</th></tr></thead><tbody><tr><td>ADCK1</td><td>NM_020421.3:c.628_633delATGT GGinsGAGTTCTT</td><td>p.Met210fs</td></tr><tr><td>AKAP3</td><td>NM_001278309.1:c.2098_2100del TCTinsCTG</td><td>p.Ser700Leu</td></tr><tr><td>ANKRD36</td><td>NM_001164315.1:c.2693_2694del ICCinsAT</td><td>p.Ala898Asp</td></tr><tr><td>ASPG</td><td>NM_001080464.2:c.283_286delTG CCinsCGCG</td><td>p.CysLeu95ArgVal</td></tr><tr><td>BAHCC1</td><td>NM_001291324.1:c.6815_6820dup p.AGGACG</td><td>p.Glu2272_Asp- 2273dup</td></tr><tr><td>C1ORF210</td><td>NM_001164829.1:c.254 A&gt; C</td><td>p.Asp85Ala</td></tr><tr><td>CCT8L2</td><td>NM_014406.4:c.551_553dupTGG</td><td>p.Val184dup</td></tr><tr><td>CEND1</td><td>NM_016564.3:c.187_192delAC CTCG</td><td>p.Thr63_Ser64del</td></tr><tr><td>CES5A</td><td>NM_001190158.1:c.470 C&gt; T</td><td>p.Ala157Val</td></tr><tr><td>CLCNKA</td><td>NM_004070.3:c.832G&gt; A</td><td>p.Asp278Asn</td></tr><tr><td>CTRC</td><td>NM_007272.2:c.419 C&gt; T</td><td>p.Ala140Val</td></tr><tr><td>LILRB5</td><td>NM_001304457.1:c.1645_1646delGAinsCC</td><td>p.Glu549Pro</td></tr><tr><td>METTL15</td><td>NM_001113528.1:c.855 C&gt; G</td><td>p.Phe285Leu</td></tr><tr><td>NTSR2</td><td>NM_012344.3:c.161_165delCGCA CinsTGCAT</td><td>p.Ala54Val</td></tr><tr><td>RPP21</td><td>NM_001199120.1:c.468_469delT CinsGA</td><td>p.Gln157Lys</td></tr><tr><td>SH3GL1</td><td>NM_003025.3:c.427delC</td><td>p.Leu143fs</td></tr><tr><td>TMEM59</td><td>NM_001305066.1:c.432_434del GCA</td><td>p.Trp144_His- 145delinsCys</td></tr><tr><td>TNIK</td><td>NM_015028.3:c.1762G&gt; T</td><td>p.Val588Phe</td></tr><tr><td>TRIM39-RPP21</td><td>NM_001199119.1:c.1491_1492del ITCinsGA</td><td>p.Gln498Lys</td></tr><tr><td>UGT2B28</td><td>NM_053039.1:c.1401_1406delGA TGTGinsCATGCC</td><td>p.Cys469Pro</td></tr><tr><td>UVSSA</td><td>NM_001317934.1:c.608T&gt; A</td><td>p.Leu203Gln</td></tr></tbody></table>

(region located upstream of the variant p.Leu143Serfs*9). Endophilin A2 expression was significantly decreased in the patient compared to his parents and two healthy controls (Fig. 1C).

## B cell Defects in the Patient

Endophilin A2-deficient mice *Sh3gl1*−/− show impairment in germinal center B cell responses and in high-affinity IgG production [8]. In-vitro B cell proliferation was diminished in the patient compared to three age-matched controls, following 3 days of stimulation with anti-CD40 (10 µg/mL) and IL-21 (50ng/mL) (Fig. 2A). In-vitro IgG production was assessed in PBMCs from the patient compared to three age-matched healthy controls, following 7 days of stimulation with anti-CD40 at 10 µg/ml and IL-21 at 50 ng/ml. This showed an abolished in-vitro production of IgG in the patient (Fig. 2B). B cell immunophenotyping in peripheral blood from the patient showed increase in naïve peripheral

B lymphocytes (CD45⁺CD19⁺CD27⁻IgD⁺), and decrease in switched memory B cells (CD45⁺CD19⁺CD27⁺IgD⁺) in the patient, compared to three healthy age-matched controls (Fig. 3A and B), indicating a defect in B cell activation.

## T-cell Proliferation in the Patient

Endophilin A2 deficiency in rodents confers protection against autoimmune arthritis, through modulation of T cell activation [16]. In-vitro T cell proliferation in response to CD3/CD28 ligation, and to Phytohemagglutinin (PHA) was modestly decreased in the patient compared to three age-matched controls (Supplementary Fig. 1).

## Discussion

Here we report a homozygous frameshift mutation in *SH3GL1* (NM_003025.3:c.427delC; p.Leu143Serfs*9) in a 14-year-old boy, issued from a consanguineous Lebanese family, and presenting since the age of 18 months with recurrent respiratory tract infections, low peripheral B cell counts and pan-hypogammaglobulinemia, with no history of opportunistic infections (Fig. 1).

*SH3GL1* encodes endophilin A2, the only member of the endophilin protein family that is expressed in hematopoietic cells [8]. Endophilins, which are classified into A and B subgroups, are evolutionary conserved proteins that contain the N-terminal BAR secondary structure (comprising three α-helices), complemented by a C-terminal SH3 domain [9]. Endophilin A modulates membrane curvature in the endocytic pathway, and is required for the FEME, a fast-acting, tubulovesicular, clathrin-independent endocytic pathway [11, 23]. FEME mediates the ligand-triggered uptake of various G-protein coupled receptors and receptor tyrosine kinases in a dynamin-dependent manner [24]. In B cells, endophilin A2 is recruited to ligand-activated BCRs through complexes formed by the BCR adaptor protein GRB2 with a possible contribution for BLNK, and regulates antigen endocytosis [8]. While endophilin A2-mediated endocytosis was shown to be dispensable for antigen presentation in B cells, it is specifically essential for providing metabolic support during B-cell growth and proliferation. This is linked to a decrease in mitochondrial respiration capacity. Endophilin A2-deficient mice *Sh3gl1*−/− show intrinsic defects in B cells, impairment in germinal center B cell responses and in the production of high-affinity IgG [8].

Interestingly, in the current report, endophilin A2 expression is significantly abolished in the patient cells compared to controls (Fig. 1C), thus confirming the pathogenicity of the p.Leu143Serfs*9 variant. On the other hand, immunoblotting for endophilin A2 using N-terminal specific antibody
A PBMCs gated on CD45+ CD19+ B cells

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

Patient

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

Control 1

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

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

Control 2

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

Control 3

B
In-vitro IgG production

<figure><img src="image_9.png" /><figcaption>**Fig. 2** Impaired in-vitro B cell proliferation and antibody production in the patient. A: Representative flow cytometry data of in-vitro B cell proliferation following stimulation with anti-CD40+IL-21 in the patient compared to age-matched controls. B: In-vitro production</figcaption></figure>

did not detect any smaller protein band in the patient com-
pared to controls, thus excluding the possible production
of a truncated form of the protein and suggesting that the
phenotype of the patient likely results from deficiency in
endophilin A2.

Similarly to Sh3gl1-/- B cells that fail to expand in
response to CD40L [8], in-vitro B cell proliferation was
diminished in the patient compared to three age-matched
controls (Fig. 2A). This was accompanied with an abolished
in-vitro production of IgG in the patient (Fig. 2B), findings
consistent with data in mice that showed a B cell-intrinsic
role of endophilin A2 and an involvement in antibody pro-
duction [8]. This is further supported by the increase in
naïve peripheral B lymphocytes and decrease in switched
memory B cells observed in the patient, compared to three
healthy age-matched controls (Fig. 3A and B), indicating a
defect in B cell activation.

On the other hand, data in Sh3gl1-/- mice showed nor-
mal early B-cell development in the bone marrow and in
the transitional populations in the spleen but a reduction
in splenic follicular and marginal zone B cells, as well as
mature B cells in lymph nodes [8]. The B-cell lymphopenia
detected in Sh3gl1-/- mice was reflected in reduced spleen
size compared with WT littermates [8]. In the current report,

of IgG in the patient compared to three healthy age-matched con-
trols. Results represent means ± S.E.M. ****p < 0.0001; by two-way
ANOVA

the defect seems to be more pronounced as total circulating
B lymphocytes counts are considerably low in our patient
(Table 1). The patient herein reported also harbors a frame-
shift variant in *ADCK1*, gene involved in the maintenance
of mitochondrial membrane structure and function. While
previous studies in Drosophila have focused on the role of
this gene in the maintenance of mitochondrial membrane
integrity, particularly in muscles, we cannot exclude its
possible implication in B and T cells’ homeostasis in our
patient. Documenting additional cases with *ADCK1* muta-
tions will be essential to elucidate whether this gene may be
also contributing to the disease in our patient.

Last but not least, immunophenotyping in the patient
revealed mild T cell lymphopenia, modestly decreased
CD8 T cell counts, with an increase in effector memory
CD4+ T cells, decrease in central memory CD4+ T cells;
decrease in central memory CD8+ T cells and increase in
CD8+ TEMRAs. This may indicate T cells’ exhaustion due
to chronic infections, or a possible role for endophilin A2 in
T cells’ development or activation. In a single experiment
we found that the proliferation of patient T cells was mod-
estly decreased in response to CD3/CD28 ligation, and to
Phytohemagglutinin (PHA) (Supplementary Fig. 1). This
partial defect in T cell proliferation is consistent with the
<figure><img src="image_10.png" /><figcaption>**Fig. 3** B-cell defects in the patient included in this study. A Representative flow cytometry data representing immune phenotyping of the patients' B lymphocytes compared to a healthy age-matched control. B Quantification of B cell subsets in the patient compared to three healthy age-matched controls. Gating on CD45⁺CD19⁺ cells, B cell</figcaption></figure>

subpopulations were defined as follows: Naïve B cells (CD27⁺IgD⁺), Unswitched memory B cells (CD27⁺IgD⁺), Switched memory B cells (CD27⁺IgD⁻), and late memory B cells (CD27⁻IgD⁻). Results represent means ± S.E.M. **p**<0.01; by unpaired T test

finding that rodents that were protected against arthritis had defective expression of SH3gl1, associated with decreased in-vitro T cell proliferation downstream of CD3/CD28, possibly affecting T cell effector functions and altering the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease [16]. Confirmatory studies in the current case were not feasible, because of the unavailability of additional samples from the patient for further investigations.

Altogether, our data suggest that endophilin A2 deficiency in humans impairs antibody production. Reporting further cases with *SH3GL1* mutations is needed to better characterize the IEI linked to endophilin A2.

**Abbreviations**

CVID Common Variable Immunodeficiency

IEI Inborn Error of Immunity

IVIG Intravenous Immunoglobulin

PAD Predominantly or Primary antibody deficiencies

PMA Phorbol Myristate Acetate

TEMRAs T effector Memory RA⁺ Cells

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s10875-024-01827-1.

**Acknowledgements** We would like to thank Dr Eileen Marie Hanna for managing the in-house NGS database. We also express our appreciation to the family, and to the clinical laboratory at the Lebanese American University Medical Center - Rizk Hospital for their cooperation throughout the study. We are thankful to the L'Oréal-UNESCO For Women in Sciences Levant young talents program.

**Author Contributions** CM, EC and AM designed the study, performed genetic and functional studies, interpreted data and wrote the manuscript. AK, SEH, SC, WF, RK, TN, contributed to technical work. RF, GL, and VD contributed to the interpretation of clinical and genetic data. IM contributed to the interpretation of clinical laboratory data. All authors approved the manuscript.

**Funding** This study was funded by the President Intramural Fund (PIRF) of the Lebanese American University (PIRF- I0003), and by the L'Oréal-UNESCO For Women in Sciences Levant young talents grant attributed to CM. Funding sources did not contribute to any of the following: study design, data collection and analysis, manuscript writing and submission.

**Data Availability** No datasets were generated or analysed during the current study.
## Declarations

**Ethics Approval and Consent to Participate** This study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Approval to conduct the study was obtained from the IRB of the Lebanese American University, Lebanon (LAU.SOM.CM1.2023.R1.1/Feb/2024). Written informed consent for analysis, and data publication was obtained from the family in compliance with national ethics regulation. Assent forms have also been obtained from the participating healthy controls, and appropriate consent forms from their parents.

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

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