Displaying One Session

E-Poster Discussion
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
E-Poster Discussion Immune dysregulation & autoimmunity

NEUROIMAGING FINDINGS IN CHILDREN WITH HEREDITARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS – REPORT FROM THE GERMAN REFERENCE CENTER

Lecture Time
13:15 - 13:22
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Immune dysregulation & autoimmunity

Abstract

Background and Aims

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome characterised by prolonged fever, hepatosplenomegaly and pancytopenia. In the hereditary form, patients may develop inflammatory CNS involvement, presenting with meningism, cranial nerve palsies and seizures, resulting in pathological neuroimaging.

Methods

We aimed to identify the most common neuroimaging findings in patients with hereditary HLH, recruited from the HLH94 and HLH2004 studies in Germany, Switzerland, and Austria.

Results

MRI was available for 37 patients (20f, 17m; median age, 1.5yrs [min, 0.1; max, 17.8]). In patients with more than one MRI, the scan with most pathological changes was selected for cross-sectional analysis. Bilateral symmetric cerebellar lesions were most common (41%, A), followed by diffuse central (38%, B), subcortical (36%) as well as periventricular (31%), cortical (31%, C) and focal subcortical (28%, D) lesions. Contrast enhancement (19%, E) and restricted diffusion (15%) were recorded. Two patients presented with imaging findings of “PRES”. Brain atrophy (F) and abnormal myelination were frequently identified and may be treatment-related. Imaging of the spine (5 patients) showed focal hyperintensity on T2w and focal contrast enhancement.pic_mri_neurohlh.png

Conclusions

Neuroimaging in patients with familial HLH is variable and non-specific, but bilateral symmetric cerebellar lesions, if present, are relatively characteristic in the clinical context.

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E-Poster Discussion Diagnostics

SPONTANEOUS SOMATIC REVERSION IN A SUBSET OF CD4+ T CELLS IN A PATIENT WITH X-LINKED HYPER IGM SYNDROME (CD40L DEFICIENCY)

Lecture Time
13:22 - 13:29
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Diagnostics

Abstract

Background and Aims

Somatic reversion of genetic defects has been observed in primary immunodeficiencies where a selective growth or survival advantage exists for the reverted cells. Somatic reversion has not been described in X-linked hyper IgM syndrome. We describe a somatic reversion in CD4+ T cells from one of two affected siblings with a novel CD40LG pathogenic variant.

Methods

Flow cytometry was used to evaluate CD40L protein expression and function on activated T cells using monoclonal antibodies and a CD40-muIg fusion construct. Sanger sequencing was performed on CD40LG gDNA and cDNA.

Results

The revertant patient presented at 9 y/o with recurrent sinopulmonary infections, low IgG, normal IgM and IgA. Unlike his brother, who lacked CD40L expression on activated T cells, the patient had two cell populations, one lacking CD40L, and a second with normal CD40L. Binding of the CD40-muIg fusion protein showed a similar pattern. Surprisingly, no pathogenic variant was found in the coding regions of the CD40LG gene but sequencing of the cDNA identified a 118 base pair insertion between exons 4 and 5. A single-nucleotide substitution (C>T) deep in the intervening intron created a functional splice donor resulting in insertion of 118-bp as a pseudo-exon between exons 4 and 5. A frameshift and premature termination resulted. The variant was hemizygous in the brother lacking CD40L expression, whereas the patient was heterozygous and had a mixed population of wild-type and mutant mRNA.

Conclusions

This case demonstrates that spontaneous reversion can be seen in patients with CD40L deficiency and can complicate diagnosis

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E-Poster Discussion Diagnostics

UNBIASED GENOMIC APPROACH TO MOLECULAR DIAGNOSIS IN PATIENTS WITH IMMUNE SYSTEM DYSFUNCTION LEADS TO UNEXPECTED MOLECULAR FINDINGS, MULTIPLE DIAGNOSES AND SECONDARY FINDINGS.

Lecture Time
13:29 - 13:36
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Diagnostics

Abstract

Background and Aims

In the past decade, dozens of new Mendelian disorders of immunity have been recognized. Yet, the genetic contributions to these disorders remain largely unelucidated. Making progress in this area requires a coordinated, systematic, and transparent approach to clinical genomics and research.

Methods

We systematically applied genomic evaluation to research participants, including clinical grade interpretation and reporting of primary and secondary findings. We collect genomic data, family medical history, and standardized phenotype. We interpret and report clinical results in the medical record in conjunction with genetic counseling and shared access to data.

Results

We have recruited 1798 participants and completed analysis for 721 individuals and finalized 342 cases. We have issued 249 (72.8%) proband reports; 111 were inconclusive and 138 had at least one molecular finding. Of these, 82 (59.4%) had a pathogenic variant, 16 (11.6%) had a likely pathogenic variant, 39 (28.3%) had a variant of uncertain clinical significance (VUS) and 1 (0.72%) had likely benign variants. Four (2.9%) cases had secondary findings including pathogenic variants in BRCA1 and BRCA2. Two (1.4%) cases had dual molecular diagnoses and 2 (1.4%) cases had a molecular diagnosis outside of “immune” phenotypes. Two (1.4%) cases had revised molecular diagnoses. Lastly, we identified several candidate genes requiring further follow up.

Conclusions

Unbiased genomic work-up yields molecular diagnoses, multiple or unexpected diagnoses, and secondary genomic findings, personalizing patient care. Process standardization, data integration, and data sharing policy facilitate research and discovery of candidate genes.

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E-Poster Discussion Other

EBV PCR IN LYMPHOCYTE SUBSET POPULATIONS AS A TOOL FOR ASSESSING CHRONIC EBV VIRAEMIA AND EBV RELATED DISEASE.

Lecture Time
13:36 - 13:43
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Other

Abstract

Background and Aims

EBV tropism in non B cells has been demonstrated to be associated with various EBV related haematological and non haematological diseases. Using cell sorting we have analysed the EBV tropism in patients with chronic EBV viremia or severe EBV related disease

Methods

37 patients with EBV viremia > 6 months and /or EBV related disease had EBV cell tropism analysed.

Results

Patients with PID, severe EBV disease and cell specific EBV tropism
Variables Present Absent Percentage (%)of patients
Underlying PID 20 17 54%
Severe EBV disease 14 23 37%
NK cell tropism 18 10 48%
T cell tropism 15 22 40%
B cell tropism 28 4 75%

20 out of the 37 patients (54%) had an underlying primary immunodeficiency. Severe EBV related pathology was seen or developed in 14 patients, 8 of whom had a T cell trophic virus and 4 patients had both T and NK cell tropism. Out of 6 patients with exclusive B cell tropism with no virus detected in T or NK cell only one had a severe EBV related disease (HLH). 18 patients with NK cell trophic virus, 8 patients had a severe EBV pathology

Conclusions

In our cohort, patients with T cell and NK cell trophic virus had a higher incidence of severe EBV related disease and persistence of viremia. Patients with an underlying PID had a higher incidence of EBV infected T cells. Cell specific EBV tropism is a useful test in risk stratification for patients presenting with persistent EBV viremia or EBV associated disease

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E-Poster Discussion Diagnostics

STIM-1 GENE MUTATIONS IN A GROUP OF PATIENTS

Lecture Time
13:43 - 13:50
Presenter
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Diagnostics

Abstract

Background and Aims

Ca release is important during lymphocyte activation. In the absence of the STIM-1,lymphocyte stimulation was impaired although the lymphocyte count was normal.In 7 patients with PID, a pathogenic variant in the STIM-1 gene has identified by targeted next generation gene sequencing.

Methods

Genomic DNA was extracted from whole blood using EZ1 DNA Kit (Qiagen).Targeted next generation sequencing (NGS) was performed using the PID v1 panel and Ion Torrent PGM sequencer with an average coverage of 100X. Variant calling and coverage analysis was performed using Ion Reporter software 5.10 (ThermoFisher).

Results

Patient Number

Age (Year/Month)

Sex

Consanguinity

Infection

Autoimmunity

Lymphoproliferation

Heredity

Mutation (Exon/Protein/cDNA)

H1

10/8

M

First degree

+

+(ectodermal dysplasia)

-

Homozygous

12

P?

c.970-1G>A/c.970-1G>A

H2

12/2

M

First degree

+

+

-

Heterozygous

11

p.(=)

WT/c.1833G>T

H3

9/7

F

-

+

+

+

Heterozygous

12

p.Pro711Ala

WT/c.2131C>G

H4

6/10

F

-

+

+

+

Heterozygous

12

p.(=)

WT/c.82C>G

H5

14/2

M

-

+(recurrent myocarditis )

-

-

Heterozygous

8

p.Ser630Phe

WT/c.1889C>T

H6

14/1

F

-

+

+

+

Heterozygous

10

p.(=)

WT/c.1299C>T

H7

10/8

M

-

+

+

+

Heterozygous

11

p.Gln507His

WT/c.1521G>C

Conclusions

The symptoms seen in our patients suggest that we should look through a large window while considering a defect and that we should examine not only the visible part of the iceberg but also underlying part of it. The data to be obtained with the development of new technologies will contribute to the clinical follow-up and treatment of patients.(Ethics Committee Decision Number: GO 15/370). Functional studies are planned.

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E-Poster Discussion Diagnostics

MOLECULAR GENETIC TESTING IN IMMUNE DISORDERS IN AZERBAIJANIAN CHILDREN

Lecture Time
13:50 - 13:57
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Diagnostics

Abstract

Background and Aims

The application of molecular genetic testing in primary immune deficiencies (PID) and other hereditary disorders allows to detect the patients with immune disability and to optimize and personalize treatment approach. According to the classification offered in last decade by many international organizations like JMF, PAGID and ESID, the diagnostic criteria of the PID are divided into different types and subgroups, mutation detection is the most reliable method of making a diagnosis. Therefore, we conducted a comprehensive molecular analysis of the patients for a definitive diagnosis.

Methods

All the patients suspected on hereditary diseases, especially PID, have to be evaluated by the disease history and wide immunological examination. In the next step, DNA sequencing and linkage analysis was performed as first-line methods in single gene disorders followed by the next generation sequencing gene panels. Karyotype analysis and FISH was applied when necessary.

Results

The molecular testing of the samples of Azerbaijanian children revealed disease associated mutations in ATM(n=5), BLNK(n=1), BTK(n=1), LRBA(n=1), IL12RB1(n=1), WAS(n=1) and HBB(n=3) genes. Deletion of the 22q11.2 segment was detected in one patient. Next generation sequencing analysis revealed variants in SHARPIN, PIK3R1, CFTR, ISG15, IRF8, DNAH1; and MTOR, CLPB8, TMC8, IL15RA genes in two patients respectively.

Conclusions

We could establish a definitive diagnosis for the patients with Louis–Bar syndrome, autosomal recessive agammaglobulinemia, Bruton's agammaglobulinemia, LRBA deficiency, complete IL12RB1 deficiency, Di George syndrome, Hyper-IgM syndrome type1, and Wiskott–Aldrich syndrome. Also we identified co-inheritance of beta-thalassemia in two patients with SCID and one patient with Louis–Bar syndrome.

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E-Poster Discussion DNA repair disorders

RADIOSENSITIVITY ANALYSIS IN PATIENTS WITH A PRIMARY IMMUNODEFICIENCY DISEASE

Lecture Time
13:57 - 14:04
Presenter
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
DNA repair disorders

Abstract

Background and Aims

Primary Immune Deficiency diseases are life-threatening genetic disorders of the immune system. A subset of PIDs is caused by mutations in genes involved in the repair of DNA double-strand breaks, by which affected patients may also be radiosensitive. For many reasons PID patients can be exposed to radiation (bone marrow transplant conditioning, radiotherapy, diagnostic imaging), but this may pose serious risks to RS subjects. Surprisingly, radiosensitivity testing is currently not included in the workup of PIDs in most European countries. This project aims to implement radiosensitivity analysis in the routine diagnostic workup for PIDs in Belgium

Methods

Two cell-cycle specific in vitro radiosensitivity assays will be included in the standard diagnostic procedures in patients with suspected PID at the Ghent University Hospital: (1) the G0 cytokinesis-block micronucleus assay (CBMN) which is about to be translated into the clinical practice, (2) the S/G2 CBMN which has been developed in our labs but which must be further optimized before translation. Both assays will be performed on peripheral blood lymphocytes of the patients. Furthermore, radiosensitivity assays will also be optimized for patients' fibroblasts. In our study design, radiosensitivity analysis will be the central core of two innovative diagnostic and therapeutic algorithms, which will include immunophentyping, direct subsequent genetic analysis and guide optimal patient care.

Results

Micronucleus analysis in lymphocytes of patients is currently ongoing and first results will be presented.

Conclusions

Inclusion of radiosensitivity testing will represent a major improvement in the timely diagnosis and management of patients affected by these life-threatening, heterogeneous and difficult-to-diagnose diseases.

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E-Poster Discussion Diagnostics

MEASUREMENT OF IMMUNOGLOBULIN G LEVELS FROM DRIED BLOOD SPOTS (IG-DBS)

Lecture Time
14:04 - 14:11
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Diagnostics

Abstract

Background and Aims

Immunoglobulin G (IgG) therapy is an effective treatment for primary immunodeficiency disease (PID) and concomitant antibody deficiencies. Currently, no option exists for home-based monitoring of IgG serum levels. Our aim was to develop an assay to measure IgG blood levels from dried blood spots (DBS).

Methods

For all tests, IgG levels in DBS eluates were directly compared to the routine method for serum IgG testing by nephelometry (BN Prospec, Siemens, Germany). A comparison was conducted of the determination of IgG serum levels in DBS eluates by ELISA and by nephelometry with instrument settings for analysis of cerebrospinal fluid IgG.

Results

ELISA and nephelometry both revealed good precision; nephelometry showed higher accuracy and was selected for further tests. The Whatman 903 DBS card demonstrated superior performance of four capillary blood collection devices assessed. The DBS inter-assay coefficient of variation (CV) was evaluated by single nephelometric measurements of replicate eluates (CV<7.5%). Intra-assay CV was assessed by replicate nephelometric measurements of a single eluate (CV=1.18%). DBS card storage stability was tested at different temperatures for several weeks, comparing IgG levels with those assayed without storage. DBS IgG levels were stable for up to one week (<10% deviation at all tested temperatures). The IgG-DBS assay was validated in 90 patients with PID. DBS nephelometry was highly correlated with serum nephelometry IgG testing (Spearman r=0.95).

Conclusions

Preliminary data suggest that patient-friendly, home-based monitoring of IgG levels from DBS could be established; further research is needed to confirm applicability in routine clinical practice.

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E-Poster Discussion Diagnostics

FLOW-CYTOMETRIC ASSAY FOR SPECIFIC CELL-MEDIATED IMMUNE-RESPONSE IN ACTIVATED WHOLE BLOOD (FASCIA) IS EMPLOYABLE IN COMBINED IMMUNODEFICIENCY (CID) DIAGNOSTICS

Lecture Time
14:11 - 14:18
Room
Station 1
Date
19.09.2019, Thursday
Session Time
13:15 - 14:20
Presentation Topic
Diagnostics

Abstract

Background and Aims

Measuring lymphocyte responses to mitogens is a key part of the diagnostics of severe combined immunodeficiency (SCID) and CID. Currently, mostly radioactive thymidine incorporation and CFSE dilution are used. Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) has been put forth as a robust and easy-to-perform option for the measurement of lymphocyte responses. Our aim was to analyze retrospectively the employability of FASCIA in the diagnostics of CID.

Methods

We included all lymphocyte stimulation tests done with FASCIA in HUSLAB between February 2015 and September 2018 in our analysis. The cohort was divided into two groups by the patients’ final diagnoses: CID (27 patients, including 7 SCID) or non-CID (159). The Kruskall-Wallis and Mann-Whitney tests were used in the analysis of the FASCIA results. The comparisons were between the FASCIA-score (mean of all mitogens) and individual mitogens. We also evaluated the sensitivity and specificity of FASCIA with the ROC-analysis.

Results

The FASCIA-score was significantly lower among the CID compared to the other patients (p< 0.001). In the ROC-analysis the AUC was 0.75 (p< 0.001) for the FASCIA-score. Of the three mitogens, PHA was best in separating patients with CID from the rest (in the ROC-analysis AUC 0.71, p = 0.001). PWM had the largest variation even among the healthy controls.

Conclusions

We conclude that FASCIA can reliably detect the CID patients from a population tested for suspected immunodeficiency. It emerges as a method with many benefits compared to tests requiring radioactive reagents or the complicated CFSE staining.

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