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Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Poster Display B Cell Biology

APPROACH TO GENETIC ANALYSIS OF NON-MONOGENIC ANTIBODY DEFICIENCY

Lecture Time
10:00 - 10:01
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
10
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Primary antibody deficiencies (PAD) are caused by developmental or functional defects of humoral immunity, resulting in increased susceptibility to bacterial and viral infections. Etiology of 40-80% of patients with PAD, the second most common type of human immune system disorders after human immunodeficiency virus infection, is yet unknown.

Methods

Monogenic patients were identified in a CVID cohort using whole exome sequencing and full-resolution MHC typing. Exome-wide polygenic scores were developed using significantly different variants and multi-variant Mendelian-randomization (MR) analyses were used to test the causality of significant genetic variants on antibody levels and susceptibility to infectious diseases.

Results

Among 83 CVID patients (44.5% females), monogenic defects were found in 40 individuals. Evaluation of the remaining patients with non-monogenic CVID showed 13 and 27 significantly associated MHC-class I and II alleles, respectively. The most significant partial haplotype linked with the non-monogenic from of CVID was W*01:01:01-DMA*01:01:01-DMB*01:03:01:02-TAP1*01:01:01 (P<0.001), where carriers had a late onset of the disease, an infections only clinical phenotype, a sporadic form of CVID, post-germinal center defects and a non-progressive form of their disease. Exclusion of monogenic diseases allowed MR analyses to identify significant genetic variants associated with bacterial infections and improved discrepancies observed in MR analyses of previous studies with low pleiotropy mainly for a lower respiratory infection, bacterial infection and Streptococcal infection.

Conclusions

The full-resolution MHC typing and polygenic scores on CVID patients showed that exclusion of monogenic forms of the disease unraveled an independent role of MHC genes and common genetic variants in the pathogenesis of CVID.

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Poster Display B Cell Biology

NOVEL FLOW CYTOMETRIC TOOLS FOR THE DISSECTION OF THE PRE-GERMINAL CENTER B CELL MATURATION PATHWAY IN COMMON VARIABLE IMMUNODEFICIENCY

Lecture Time
10:01 - 10:02
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
11
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Common Variable Immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency. A significant proportion of patients present decreased number of circulating B cells, and defective or deregulated pre-germinal center B cell compartment.

We aim to precisely trace the maturation pathway of the pre-germinal center compartment in CVID patients, to identify if a deregulated or derailed maturation could already indicate an impaired early development in bone marrow, or suggest alterations in peripheral B cell homeostasis in a proportion of patients.

Methods

A total of 100 CVID patients and 56 HD were studied with standardized protocols within the collaborative and multicenter frame of the PID-group of the EuroFlow consortium. Patients samples and clinical data were collected at 7 different sites.

The distribution of distinct B-cell subsets were analyzed by flow cytometry, with the EuroFlow 8-color pre GC B-cell tube. CD19+ B-cells were subclassified into 11 different subpopulations.

Results

We created a normal maturation B cell pathway focused in the pre GC B cell compartment of HD with the maturation tool of the Infinicyt software. We performed the same analysis to a cohort of 100 CVID patients and plotted the individual results to the normal, in order to detect aberrant populations in preGC in CVID that may have followed a diverse maturation pathway, and identify aberrant expression of markers in CVID patients.

Conclusions

We sought for correlations among alterations in the preGC compartment (absolute counts and expression markers) with clinical data records looking for potential implications of phenotypic aberrancies and clinical complications.

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Poster Display B Cell Biology

IMMUNOGLOBULIN G SUBCLASS AND SPECIFIC POLYSACCHARIDE ANTIBODY DEFICIENCY IN CHILDREN AND ADULTS: A CLINICAL AND IMMUNOLOGICAL PROFILE IN A TERTIARY COHORT

Lecture Time
10:02 - 10:03
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
12
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Immunoglobulin(Ig)G-subclass deficiency (IgGSD) and specific polysaccharide antibody deficiency (SPAD) are two common antibody deficiencies, mainly associated with recurrent respiratory tract infections (RTI). The clinical variability and effect of therapy in a large cohort is not well studied.

Methods

A retrospective observational study of children and adults with IgG2SD and/or IgG3SD and/or SPAD and normal total IgG.

Results

In total, 105 patients (age 2y-79y (mean 24y), 53% pediatric, 44% male) were included. To our knowledge, this is the largest cohort of IgGSD and/or SPAD patients studied. Isolated IgG3SD was both in children (16/56;29%) and adults (30/49;61%) most prevalent. Of all IgG2SD(+/-SPAD) cases, 85% were found in children (22/26). In total, isolated SPAD was less frequent (16/105;15%) than IgGSD+SPAD (21/105;20%).

Recurrent upper and lower RTI (76-82% and 38-57% respectively) were most frequently observed, similar in all groups. Gastrointestinal infections (42%;P=0.017) and fatigue (45%;P=0.005) were associated with IgG3SD. Bacterial skin infections (38%;P=0.024) were frequently observed in SPAD. Autoimmunity (25%;P=0.001), lymphadenopathy (71%;P=0.002) and fatigue (59%;P<0.001) were significantly common in adults compared to children. Autoimmunity was not associated with aberrant B-cell maturation, only present in 2.8%. IgG2SD patients (69%;P=0.046) and children (73%;P<0.001) were hospitalized more.

Monocytopenia was repeatedly observed (32/105;30%), predominantly in IgG2SD (15/31;48%) and persistent, without effect of Ig replacement therapy. In patients with Ig replacement, bacterial infection rate reduced significantly in 82%, although low IgG3 persisted.

Conclusions

RTI remain the hallmark presentation in IgGSD and/or SPAD. Our cohort revealed remarkable findings, such as frequent autoimmunity and monocytopenia. Long-term multi-centre studies are needed to better characterize these patients.

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Poster Display B Cell Biology

SERUM B CELL MATURATION ANTIGEN (BCMA) LEVELS DIFFERENTIATE PRIMARY ANTIBODY DEFICIENCIES

Lecture Time
10:03 - 10:04
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
14
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Primary antibody deficiencies (PAD) are the most prevalent primary immunodeficiencies. Severer forms of PAD, common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA), require immunoglobulin replacement therapy (IRT) and may lead to serious complications. Differentiating severe PAD from hypogammaglobulinemia not requiring IRT can involve prolonged evaluations and treatment discontinuation. Severe PAD is defined by plasma cell deficiency, but this requires biopsy to establish. Serum B cell maturation antigen (sBCMA) is elevated in multiple myeloma, but levels are reduced among myeloma patients in complete remission who have hypogammaglobulinemia. We measured sBCMA levels in 165 subjects to determine whether it differentiates the severe PAD, CVID and XLA, from less severe forms not requiring IRT and those without PAD.

Methods

sBCMA, B-cells, and tissue plasma cells were measured among subjects with and without PAD, and correlated to clinical and laboratory data.

Results

Subjects with IgG < 600 mg/dL had reduced sBCMA levels compared with PAD with IgG ≥ 600 and non-PAD controls. sBCMA was lower in CVID and XLA compared to IgA or IgG deficiency and non-PAD controls. sBCMA correlated with gastrointestinal plasma cells. sBCMA < 15 ng/mL had 97% positive predictive value for CVID or XLA, while 25 ng/mL or more had 88% negative predictive value.

Conclusions

sBCMA is profoundly reduced in severe PAD, including CVID, XLA and subjects with IgG < 600 mg/dL. sBCMA measurement has potential to augment clinical evaluation of PAD. Prospective studies are needed to evaluate sBCMA for new PAD diagnosis and determining necessity of IRT.

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Poster Display B Cell Biology

PROTEIN LOSING ENTEROPATHY AS A COMPLICATION AND/OR DIFFERENTIAL DIAGNOSIS OF COMMON VARIABLE IMMUNODEFICIENCY

Lecture Time
10:04 - 10:05
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
178
Presentation Topic
B Cell Biology

Abstract

Background and Aims

As protein-losing enteropathy (PLE) can lead to hypogammaglobulinemia and lymphopenia, and since common variable immunodeficiency (CVID) is associated with digestive complications, we wondered if: 1/PLE could occur during the course of CVID; and 2/PLE was associated with specific immunological anomalies that differed from CVID.

Methods

Patients were classified in 3 groups: CVID+PLE+ (n=5), CVID+PLE- (n=21), PLE+CVID- (n=12). PLE was diagnosed using α1-antitrypsin faecal clearance or Gordon-Waldmann test. Immunoglobulin (Ig) A, G and M levels, and naïve/memory B and T cell subsets were compared between each group.

Results

Most CVID+PLE+ patients had multiple causes of PLE: inflammatory bowel disease (3/5), nodular regenerative hyperplasia (2/5), villous atrophy (1/5), giardiasis (2/5). Compared to the CVID+PLE- group, CIVD+PLE+ patients had lower IgM levels (0.04±0.0045 vs 0.53±1.5g/l, p<0.001) and IgG substitution efficiency index (36±6.2 vs 81±26, p=0.002); but similar IgA and pre-substitution IgG levels, B and T cells counts and subset distribution. Compared to the CVID+ PLE- group, PLE+ CVID- patients did not develop infectious complications; had higher serum IgA (1.24±0.66g/l vs 0.13±0.12, p<0.001), IgG (3.8±1.7g/l vs 1.9±1.4, p=0.007) and switched memory B cells (8.5±6.2 vs 2.8±5.6%, p=0.001) levels; lower naïve CD4 (12±9.3 vs 33±22%, p=0.004) and naïve CD8 (7.3±9.9 vs 38±20%, p<0.001) T cells levels; and similar IgM, naïve and marginal zone B cells levels.

Conclusions

PLE can occur during CVID and requires higher Ig replacement therapy dosing. PLE can also mimic CVID and is associated with milder immunological abnormalities, notably mildly decreased to normal serum IgA levels.

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Poster Display B Cell Biology

DOCK11 IS A CANDIDATE GENE FOR SELECTIVE IGM IMMUNODEFICIENCY (SIGMID)

Lecture Time
10:05 - 10:06
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
15
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Selective IgM immunodeficiency (sIgMID) represents a poorly understood dysgammaglobulinemia that is characterized by persistently low serum IgM levels (≤40mg/dl) and normal IgA and IgG levels. Patients with sIgMID often suffer from recurrent and/or severe infections, while less common manifestations include fibromyalgia, arthralgia, fatigue and autoimmune disorders. In a previous study we found altered B cell subsets, diminished IgM expression and profound defects in B-cell differentiation. To better understand disease pathology we aim to elucidate the genetic origin of sIgMID.

Methods

For our genetic analyses we apply whole-exome sequencing to search for sIgMID disease mutations which we follow-up and seek to validate experimentally, for example by gene editing.

Results

So far, we identified 5 private missense variants in the gene Dedicator of Cytokinesis 11 (DOCK11) among 12 sIgMID patients. At least in one case the variant seems to affect activation of downstream proteins. In contrast, in a cohort of 100 CVID patients no private missense or nonsense DOCK11 variants were detected.

Conclusions

We found strong evidence that DOCK11 causes sIgMID. In our ongoing experiments we study the function of our DOCK11 candidate variants in B cells by applying CRISPR/Cas9-gene editing in LCLs to correct the variants back to wild type.

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Poster Display B Cell Biology

DYNAMIC IN VITRO MODELING OF HUMAN B CELL DEVELOPMENT DISSECTS EARLY DEVELOPMENTAL DEFECTS IN PRIMARY ANTIBODY DEFICIENCY.

Lecture Time
10:06 - 10:07
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
16
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Bone marrow (BM) analysis shows that 20% of CVID patients harbor a defect at an early stage of B-cell development. The aim of this work is to study the mechanisms of the developmental block observed in a subgroup of CVID patients with increased risk of malignancy.

Methods

We set up a feeder-free cultivation system, that in healthy donors leads to the development of common lymphocyte progenitors (CLP) until the stage of IgM+ Immature B-cells. BM-CD34+ cells are expanded in the presence of a cytokine cocktail for two weeks and then cells are cultivated in cytokine-free medium up to day 49 and analyzed weekly by flow cytometry.

Results

CD34+ cells from 15 CVID patients, 9 of which presented a block at early stages of development, and 2 Btk-deficient patients were tested in vitro. As expected CD34+ cells from patients with normal B-cell development in vivo developed into lymphocyte progenitors until the stage of IgM+ Immature-B cells, while in Btk-deficient patients the development arrested at Pre-B-cell stage. Among CVID patients presenting a block in development, 3 could not reach the immature B-cell stage, while 6 could develop in vitro until the stage of IgM+ B cells, even though 2 of the latter patients presented a progressive exhaustion of the CLP compartment.

Conclusions

We identified four groups: (1) with intrinsic B-cell defect, (2) with possible defect in the BM microenvironment impairing early stages of B-cell development (3) with defect in repopulation of CLP compartment and (4) with normal B-cell development.

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Poster Display B Cell Biology

A PATİENT WİTH GERMLİNE HETEROZYGOUS MİSSENSE IKZF1 MUTATİON

Lecture Time
10:07 - 10:08
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
17
Presentation Topic
B Cell Biology

Abstract

Background and Aims

IKAROS, a transcription factor encoded by IKZF1, is expressed during hematopoiesis that is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. Herein, we report a patient with common variable immunodeficiency associated with germline missense IKZF1 heterozygous mutation.

Methods

The targeted new generation sequencing PID V1 panel was used.

Results

Case Report:

A 20-year-old male patient who was born to non-consanguineous parents presented with recurrent bacterial upper and lower respiratory tract infection, anemia and chronic diarrhea. Physical examination was normal. Laboratory tests revealed anemia, hypogammaglobulinemia and B cell lymphopenia. In B cell subsets memory B cell, switch memory B cell, marginal zone memory B cell were low. Vaccine responses and isohemagglutinin tests were poor. Colonoskopy was perfermed and revealed as inflammatory bowel disease. The genetic analysis revealed germline missense IKZF1 heterozygous mutation affecting amino acid T510A located in the Zn_C2H2 domain of IKZF1.

Conclusions

Germline heterozygous IKZF1 mutations cause hypogammaglobulinemia, hematologic abnormalities, including B-cell defect; and autoinflammatory diseases.

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Poster Display B Cell Biology

ACTIVATION-INDUCED CYTIDINE DEAMINASE MUTATION IN A PATIENTS WITH HYPER-IGM SYNDROME

Lecture Time
10:08 - 10:09
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
13
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Introduction

Activation induced cytidine deaminase (AID) is an essential enzyme for class switch recombinationand somatic hypermutation. We herein report a patient who had AID gene defect and is diagnosed as the autosomal recessive form of hyper-IgM syndrome (HIGM) type 2.

Methods

Case report

Results

A 8-year-old male patient was referred to our hospital for further evaluation of low immunoglobulin levels. The patient was the second child of the family with neither family history of immunodeficiency nor serious infections/hospitalizations up to 3 years of age. Parents were non-consanguineous and healthy. In the third year of life he had begun to suffer from recurrent otitis media and papular skin lesions. Physical examination on admission showed enlarged (1.5x1.5cm) cervical lymph nodes. Laboratory tests revealed hemoglobin levels of 13.7 g/dL, a white blood cell count of 10500/mm3, platelet count of 255000/mm3, there was not any abnormal findings in liver and renal function tests. He had high IgM, while he had low IgA, IgE and IgG (IgM 261 mg/dL, IgA <7 mg/dL, IgE <1,00 UI/mL, and IgG <33,3 mg/dL). Lymphocyte subsets, CH50, C3, and C4 levels, NBT were normal. Based on clinical/laboratory findings, the initial diagnosis was HIGM sydrome and intravenous immunoglobulin (IVIG) replacement therapy (0.4 g/kg) was started monthly. In the follow-up, we performed “next generation sequencing (NGS)” analysis for primary immunodeficiency diseases, anda homozygous c.70C>T (p.R24W) mutation was found in AID gene.

Conclusions

Conclusion

HIGM2 should be considered in patients presenting with recurrent infections. Early diagnosis and treatment will decrease the risks of complication.

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