Background and Aims

Ataxia-telangiectasia (AT) is a neurodegenerative disorder caused by mutations in the gene encoding the ATM kinase, a key regulator of the DNA damage response (DDR) pathway. Susceptibility to cancer and pulmonary infections dramatically reduce life expectancy. Over 60% of AT patients show variable immunodeficiency, with progressive B- and T-cell lymphopenia and abnormal immunoglobulin levels. Here we studied the NK cell compartment of AT patients, with a special focus on the NKG2D activating receptor that potently triggers cytotoxicity upon engagement by ligands (NKG2DL) induced via the DDR pathway on infected or transformed cells.

Methods

In four patients (AT#1-4), NK cell phenotype and function and NKG2DL expression on various cell types were analyzed by flow cytometry. Plasma soluble NKG2DLs (sNKG2DLs) were measured by ELISA. NKG2D expression was evaluated by western blotting and qRT-PCR in NK cells of AT#1-2 patients.

Results

NK cells had normal frequencies but were skewed towards the CD56^neg anergic subset in AT#1-2, with AT#2 also showing impaired NK-cell cytotoxicity. All patients showed down-regulation of at least one NK-cell activating receptor (NKG2D, NKp46, DNAM-1). When analyzed, NKG2D was reduced at the protein but not at the mRNA level and resulted in lower NKG2D-mediated cytotoxicity. In all patients, both basal and induced expression of NKG2Ls was increased on B and T lymphocytes and at least one sNKG2DL accumulated in AT plasma.

Conclusions

NK cells are disturbed in AT patients. Alteration of the NKG2D/NKG2DL axis found herein may contribute to infection susceptibility and development of lymphoproliferative disorders or malignancies.

Background and Aims

Leukocyte adhesion deficiency (LAD) is a defect of cellular adhesion molecules with deficiency of various glycoproteins including LFA-1 resulting in a combined immunodeficiency disorder with recurrent bacterial and fungal infections. The aim of our study is to describe LAD’s clinical features.

Methods

From 1998 through 2018, 18 patients (from 12 families and 11 consanguineous marriage) were enrolled. Each of them had a severe (<1%: n=15) or moderate (1-30%: n=3) decreased expression of LFA-1 and benefited from chemoprophylaxis with Cotrimoxazole and Itraconazole. Clinical data was retrospectively analyzed.

Results

Sex ratio was 0,8. Mean age was 29 days [2;180] at first symptoms and 7,6 years [0,16;31] at diagnosis. First signs included delayed separation of the umbilical cord (n=13), omphalitis (n=12), oral thrush (n=11) and sepsis (n=10). The most common infections were recurrent skin infections (n=16), ENT infections (n=7), pneumonia (n=7), urinary tract infections (n=5) and gastrointestinal infections (n=5). The main infectious agents were enterobacteriaceae (n=10), staphylococcus (n=9), pseudomonas aeruginosa (n=9), candida (n=5) and corynebacterium (n=4). All patients had marked neutrophilic leukocytosis with mean neutrophilis count at 46323 cells/mm³. Four patients were lost to follow-up. Issue was fatal in 7 cases (mean age at death= 6,5 years). Five patients had allogeneic hematopoietic stem cell transplant (HSCT), 2 of whom were cured.

Conclusions

LAD is usually fatal before 2 years of age. Moderate cases can live longer with appropriate antimicrobial therapy. Those patients with successful HSCT can have a better quality of life.

Background and Aims

Toll-like receptor 3 (TLR3) binds double stranded RNA which via TRAF3 and TBK1 leads to IRF3 phosphorylation and induction of a type I IFN response. Defects in TLR3 have been described as genetic etiology of Herpes Simplex Virus encephalitis (HSE) , with variable penetrance.

Methods

We describe here a patient with TLR3 mutation (p.Pro554Ser) who presented with several severe viral infections including severe Influenza A virus (IAV), but no HSV encephalitis.

Results

Patient is a girl from Belgian non-consanguineous parents. She presented at 5 weeks of age a severe Influenza A infection that required non-invasive ventilation and admission to pediatric intensive care (PICU). 2 months later, she developed a severe bronchiolitis due to Respiratory Syncytial Virus (RSV) and required again non-invasive ventilation at PICU. She was furthermore hospitalized between 1 and 2 times a month for respiratory or gastro-intestinal disease (including Rotavirus infection despite immunization). At the age of 6 months old, hypogammaglobulinemia was found and immunoglobulins substitution was started. Infection frequency slowly decreased. She is now 3.5 years old, alive and well. A NGS panel for immune deficiencies was started and surprisingly, returned a heterozygous TLR3 mutation (p.Pro554Ser), reported pathogenic in the context of HSE. WES did not reveal alternative etiologies for the observed phenotype.

Conclusions

We here report a pathogenic TLR3 (p.Pro554Ser) mutation in a patient with severe flu and other viral infections, but not HSE. This broadens the phenotype observed in patients with this TLR3 mutation.

Background and Aims

Coccidioides spp are pathogenic, dimorphic fungi endemic in the desert soils of the Southwestern United States. While 2/3 of those infected never come to medical attention, ~1/3 develop protracted pneumonia; less than 1% of those infected disseminate. Epidemiologic studies have demonstrated increased dissemination rates among individuals of African-American, Hispanic and Filipino descent suggesting underlying genetic susceptibility. Previously we identified causative IFNGR1, IL12RB1, STAT1 and STAT3 mutations in multiple disseminated coccidioidomycosis patients (DCM), highlighting the IFNG-IL12/23 pathway as crucial to control infection.

Methods

To expand on these findings, whole exome sequencing was performed on 49 DCM and 44 contained-pulmonary patients (pCocci).

Results

DCM had more variants with population frequency less than 10% than pCocci (p=0.0345). Comparing individual gene burdens in DCM and pCocci, we identified 310 genes significantly increased in DCM, including a subset related to iron homeostasis. Targeted gene list analysis of macrophage response genes, IFNG-IL12/23 pathway, IL17 and Dectin-1 signaling and iron homeostasis (359 genes) found more variants in DCM than pCocci patients (p=0.0122) and DCM patients had a higher burden of deleterious (CADD>20) variants than pCocci (p<0.0001). Further, patterns appear in DCM specific gene sets such as STATs, IL12, IL17 or Dectin-1 signaling, suggesting that these pathways affect dissemination. In DCM variants STAT3 (p.V461L) and DECTIN-1 (p.R238*) were over-represented compared to ExAC.

Conclusions

In conclusion, WES demonstrated distinct genetic burdens in DCM vs pCocci, implicating innate immune pathways as well as iron homeostasis. These data deepen our understanding of risk factors of disseminated coccidioidomycosis.

Background and Aims

In Japan, children under age 1 year are routinely vaccinated against Bacillus-Calmette Guérin (BCG). BCG vaccination is contraindicated in children with a primary immunodeficiency disease such as chronic granulomatous disease (CGD), which is characterized by a defect in the ability of phagocytes to form reactive oxygen species against bacteria, fungi, and BCG. Herein, we examined the frequency of BCG infection following a BCG vaccination and the therapeutic effect of antitubercular drugs in patients with CGD.

Methods

The present analysis was based on a retrospective medical record review of 42 CGD patients over the last 16 years. All treatments were given after obtaining the patients’ informed consent.

Results

Twenty-nine patients with CGD received their BCG vaccination at a clinic or hospital. Three of nine patients with a family history of CGD received a BCG vaccination inadvertently. Following vaccination, they experienced a BCG infection, including lymphadenitis (62%), skin ulcers (14%), pneumonia (10%), disseminated disease (6.8%), and dermatitis (3.4%). Of 21 patients with CGD who experienced a BCG infection, 15 patients received antitubercular drugs, including isoniazid, rifampicin, streptomycin, and clarithromycin. None of the patients experienced a life-threatening BCG infection in this study.

Conclusions

Although antitubercular drugs were effective even in CGD patients with a BCG infection, the lesions caused by the infection did not completely resolve in all the patients. To avoid administering a BCG vaccination to patients with CGD, educating both the patients and their family on the topic, detailed history-taking prior to vaccination, and newborn screening to find suspicious patients with CGD are required.

Background and Aims

Gain of Function(GOF) of STAT1 is a recent cause of PID with autosomal dominant inheritance pattern (AD). This genetic defect is accompanied by autoimmune disorders (37%), complications associated with infections (11-21%), vasculopathies (6%) and malignancy (6%). Fungal infections has a penetrance of 90% in the first decade and is usually the first sign of the disease. We presente the case of a STAT 1 GOF defect with an atypical presentation.

Methods

54 year old female with positive family history of CMC. She was initially diagnosed with diabetes mellitus (DM) 1 at 13 years. at age 20, repetitive nonivasive fungal infections (GI tract, ungueal). Candida spp. was isolated and esophageal stenosis was noted. Due to familiy history of CMC with AD pattern, sequenciation was done, confirming the diagnosis. During followup, she developed seronegative arthritis and cylindrical bronchiectases. Currently the patient is on prophylactic antifungal agents with good response and has yearly visitations to gastroenterology and dermatology where active search for skin and mucosal neoplasia is performed.

Results

We present this case due to its unusual presentation and its associated complications. Our female debuts with autoimmunity (DM1), presenting after its debut with late onset CMC. She continues to present unusual associations; esophageal stenosis at diagnosis and finally seronegative arthritis and cylindrical bronchiectases on both lower lobes at follow-up. No malignancy has been detected so far.

Conclusions

In STAT 1 GOF, it is important to detect associated complications since these associations have a tremendous impact on the prognosis of this cohort.

Background and Aims

Mendelian susceptibility to mycobacterial disease (MSMD) due to inborn errors of IFN-γ immunity.is a rare primary immunodeficiency, triggered by non-tuberculous mycobacteria or Bacillus Calmette-Guérin (BCG) vaccines.

Methods

Nine patients (7 males, 2 females) diagnosed as Interleukin-12/Interferon-gamma pathway defects were included in the study.

Results

The diagnosis were IFN-γR1 complete defect (n = 1), IFN-γR2 partial defect (n=3) and IL-12Rβ1 defect (n=5). The rate of consanguineous marriage was 78% and the primary immunodeficiency family history was 56%. BCG vaccine was applied to all cases. The presentation symptoms were recurrent respiratory infections (100%), lymphadenitis (78%), chronic diarrhea (44%) and rash (11%). On physical examination, pathological findings were pulmonary involvement (44%), lymphadenitis (78%), hepatosplenomegaly (78%), generalized lymphoproliferation (44%), eczema (11%) and edema (11%). Laboratory tests showed leukocytosis, hypergammaglobulinemia and elevated erythrocyte sedimentation rate. M. Bovis, M. Avium intracellulare, M. Fortuitum and Salmonella were isolated from lymph nodes, bone marrow and liver. Leukocytoclastic vasculitis was observed in 2 cases and granulomatous dermatitis in 1 case. Splenectomy was performed in two patients because of hypersplenism. One case with IFN-γR1 complete defect died after hematopoietic stem cell transplantation and 1 patient with IL12-Rβ1 defect died due to sepsis at the age of 17 years. Four patients underwent regular recombinant IFN-γ treatment. One patient who had symptoms of Hyper IgE syndrome on admission found to have IL12-Rβ1 defect and benefited from regular IVIG therapy.

Conclusions

IL12/IFN-γ/IL23 defects should be considered in patients with BCG complications and non-tuberculous mycobacterial infection.Early hematopoietic stem cell transplantation should be performed in IFN-γR complete defects before the development of complications.

Background and Aims

IRAK-4 is an active kinase associated with the IL1 receptor. It is involved in the signaling pathway of the cellular activation in response to Toll like receptor (TLR) agonists and the IL-1/IL-18 superfamily. Irak-4 deficiency is an autosomal recessive defect of innate immunity. We report a 17-year old female patient with recurrent infections and numerous hospitalizations since the age of 3 years. At the age of 7 years she was investigated for recurrent bacterial infections affecting the skin (Staphylococcus aureus) and the respiratory tract such as sinusitis, otitis media and mastoiditis (S. pneumoniae). She was also diagnosed with intellectual disability within autism spectrum, obesity and hypothyroidism. Her immunological investigation was normal except low immunoglobulin levels (IgG=717mg/dl, IgA=26,60mg/dl, IgM=50,80mg/dl). A prophylactic administration of immunoglobulins was started and lasted 5 years resulting in significant reduction of infections. Since the age of 12 years she is no longer suffering from infections and her immunoglobulin levels are normal.

Methods

Whole exome sequencing was performed on the patient. DNA-analysis was done using Otogenetics Corporation (USA). IRAK4-gene expression was assessed with qRT-PCR.

Results

Whole exome sequencing analysis revealed a heterozygous variant in the patient: c.823delT (p.S275fs*13) in IRAK-4 gene.

Conclusions

IRAK-4 should be considered among patients with unclassified primary immunodeficiency. The diagnosis is confirmed by IRAK-4 gene sequencing. Clinical outcome improves with age. Prophylactic treatment with immunoglobulins is recommended until the teenage years.

Background and Aims

Chronic granulomatous disease (CGD) is a rare congenital disorder of the innate immune system. CGD is caused by a defect in any one of four components of the NADPH oxidase, i.e. gp91phox, p22phox, p47phox or p67phox. Most CGD patients (~65%) from Western populations have a mutation in CYBB coding gp91phox, and some (~30%) have a mutation in NCF1. We screened X linked CGD gene to detect mutation in this male patient.

Methods

We have used flow cytometry for functional diagnosis and subgroup identification of X-CGD case whose mother is a carrier having bimodal histogram pattern in DHR assay and p22 intracelulary subgroup analysis, since gp91phox and p22 coexisting. To detect mutation we used next generation sequencing of CYBB gene mutations.

Results

The 5 year old male patient suffered due to recurrent necrotising pneumonia and widespread lypmhadenopathy after routinely applied BCG vaccination. The clinical features signified immun deficiency in particular CGD, respectively. DHR analysis showed no oxidase activitiy in neutrophils but surprisingly normal activity in eosinophils. Mother's DHR showed bimodal pattern. In total being a male and mother is a carrier we suspected CYBB gene. NGS resulted as a novel mutation in the promoter region c.-65C>G.

Conclusions

X-CGD patients with promotor mutations in CYBB is rare.NGS is a good method to detect mutations not in the exons.

The clinical phenotypes can vary.Promoter mutations are assumed to have with milder phenotype and late diagnose but our case is contradictory. Although eosinophils have almost normal oxidase activiy it is not enough for effective microbial killing.

Background and Aims

Leukocyte adhesion deficiency is a rare neutrophil disorder. Neutrophils can not migrate the inflammation site due to defect in the adhesion molecules; LFA-1 (CD11a/CD18; α_Lβ₂), Mac-1 (CD11b/CD18; α_Mβ₂), and p150/95 (CD11c/CD18; α_Xβ₂) , respectively. Leucocytosis, lack of pus formation, recurrent infections and late seperation of umbilical cord are the main characteristics of the LAD type I.LAD I is due to mutations in a gene ITBG2 that encodes CD18,the β2 integrin subunit of the heterodimers LFA-1, Mac-1 (CR3) and p150,95. Here we present a case with complete lack of CD18 but still has some pus formation and milder phenotype.

Methods

Mutation detected with Sanger method on ITBG2 gene. Facs analysis was done for adhesion molecules :CD18 CD11b CD11c for gating neutrophils:CD45 .

Results

Patient was born into a consanguinous parents who has lost previous child due to infection. Until At 7 months old patient had recurrent infections including purulent otitis media and pneumonia. CBC count showed WBC levels are high than normal(41,060) repeatedly. Through suspicion of LAD, FACS analysis detected CD11b and Cd18 absence in neutrophils. Genetical analysis also revealed C305_306delAA mutation on ITGB2 gene. Even though the mutation is damage causing and certainly complete lack of integrin molecules ; unexpectedly Patient has pus formation and milder clinical phenotype.

Conclusions

In conclusion, we reported an atypical presentation of LAD type 1 with complete lack of CD18 but still some pus formation controversially. During clinical follow up the patient had intraabdominal abcess then operated and still receiving antibiotic prophilaxis.HSCT is planned for complete recovery.

Background and Aims

Chronic Granulomatosis Disease (CGD) is a rare primary immunodeficiency disorder of bacteria and fungi that cannot be killed by neutrophils and is observed at 100-200 thousand of births. The mutation of one of the oxidase proteins (p22-phox, p47-phox, p67-phox, p40 phox) which constitutes the leucocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme structure leads to autosomal recessive CGD. The mutation of gp91 phox encoded by the CYBB gene causes X-linked CGD.

Methods

Functional study of phagocytic cells by DHR assay with PMA stimulation showed a stimulation index (SI) 1.4 fold, which is specific for CGD. MPO expression was in the normal range at cytometry. This known mutation was checked by Sanger sequencing. In the patient’s DNA a homozygous nonsense c.229C>T mutation was found in NCF2 gene (OMIM, 608515), resulting in p.[Arg77Ter] stop codon formation, which causes loss of p67-^phox protein formation and causing AR-CGD.

Results

In our studies on the diagnosis of CGD, we observed that patients come from a town, which has a population of 50 thousand and 125 km away from Kayseri City. So far, we have identified 8 patients with GGD in the region and 3 of these patients were lost, 3 of them were bone marrow transplanted, and all of them are well now.

Conclusions

A similar event has been reported in the literature on the presence of a mutation in the CYBA gene which causes AR-CGD in Korea-jeju island.We assumed that there might be an ancestral relation between these families.

Background and Aims

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disorder caused by defect in expression or function of CD18, a β2 subunit of integrins expressed on surface of neutrophils. This is characterized by early onset recurrent, life- threatening bacterial infections associated with who do not receive hematopoietic stem cell transplant.

Methods

Clinical and laboratory features of cases of LAD diagnosed single tertiary centre were retrieved and analysed. Twenty one children amongst these were diagnosed to have LAD type 1. Clinical details and laboratory investigations were analyzed. Diagnosis of LAD 1 was made on basis CD18 expression by flow cytometry.

Results

Mean age of presentation of LAD was 3.3 years (20 days – 15 years), (11 boys, 10 girls). Delayed cord fall (beyond day 15) was noted in 9, while omphalitis was present in 12 patients. Five had history of consanguinity. Two had amyloidosis and Budd-Chiari syndrome respectively. One patient had pyoderma gangrenosum, which was treated immunosuppresents. One patient presented with palatal perforation at the age of 1 month. One patient had umbilical-enteric fistula with non healing ulcers over abdomen. Mean percentage of gated neutrophils showing CD18 was markedly decreased in patients 0.76% (0-0.96%) compared to controls 96.1-100%. Genetic mutation in ITGB2 gene was confirmed in 14 patients. Of these 14 patients, 4 had p.Arg624Ter variant. Fifteen of these children have succumbed to infections, one is doing well on post HSCT.

Conclusions

Children with LAD have a high mortality in resource-limited settings in absence of readily available facilities for HSCT.

Background and Aims

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by susceptibility to bacterial and fungal infections resulting from the inadequacy of phagocytic leucocytes to produce reactive oxygen radicals. CGD is a genetically heterogeneous disease with an X-linked recessive (XR-CGD) form caused by mutations in the CYBB (OMIM #300481) gene encoding the gp91(phox) protein, and an autosomal recessive (AR-CGD) form caused by mutations in the CYBA (OMIM #608508), NCF1 (OMIM #608512), NCF2 (OMIM #608515) and NCF4 (OMIM #601488) genes encoding p22(phox), p47(phox), p67(phox) and p40(phox), respectively. The genetic mutation of one of the cytosolic p47phox/p67phox proteins and membrane-bound gp91phox/p22phox proteins, which constitutes the NADPH oxidase enzyme complex, causes the disease. In this study, we evaluated the clinical, laboratory and genetic findings and the prognostic effects of molecular inheritance of our 24 CGD cases (14 XR, 10 autosomal recessive-AR).

Methods

Consanguinity (three XR and all AR cases) showed statistically significant relationship with the type of hereditary inheritance (p<0.001).

Results

83% patients had an infection since early infancy. The mean age of initiation of symptoms was earlier in XR cases, and 78% patients had respiratory tract infections. Bone marrow transplantation was performed in five XR cases (two ex) and four AR (one ex) cases. Three of nine XR and two of six AR cases deceased on medical follow-up.

Conclusions

In countries especially with high consanguinity rates, the early diagnosis for appropriate prophylactic treatment of CGD is quietly important to avoid from recurrent severe infections, early death and fatal complications of late transplantation.

Background and Aims

Severe and recurrent infections with Herpes simplex virus type I (HSV-1) are observed in a large group of patients treated in immunology facilities, yet the pathomechanism of these infections remain largely unknown. The most important cells controlling Herpes infections are NK cells, and recent findings indicate an important role of microRNA (miRNA) regulation of gene expression by in development and function of these cells. That is why, the aim of the study was to determine a role of miRNA in severe and/or recurrent infections with HSV-1.

Methods

The miRNA expression profile was determined in NK cells isolated from patient and control subjects using TaqMan real-time PCR. Currently, the role of selected miRNAs, upregulated in a group of patients, in gene expression involved in antiviral response of NK cells is being investigated.

Results

As a result, we detected four miRNA: miR-27b, miR-199b, miR-369-3p, and miR-491-3p, which expression was significantly elevated in a group of patients, in comparison to healthy controls. These miRNAs have been described previously i.e. to attenuate inflammatory response on monocytes (miR-199b) or take part in the development of neurological disorders during other virus of Herpes family – cytomegalovirus (miR-27b), yet their function was not studied in NK cells. The role of selected miRNAs in regulation of antiviral response of NK cells should be presented at the poster.

Conclusions

At this stage, it is not clear whether the observed alteration of the expression of microRNAs is a cause of observed disturbance of antiviral response or a result of prolonged viral infection.

Background and Aims

TLR3 and UNC93B are proteins necessary to induce type 1 IFN and proinflammatory cytokines, crucial in anti-viral immunity. AD-TLR3 deficiency has been identified as a disorder of HSV-1 encephalitis, not by EBV. Moreover, EBV–encoded small RNA induces signaling from TLR3.

Aim:To report a patient with genetic variations in TLR3 pathway with uncommon symptoms.

Methods

Review medical record

Results

An Argentinean 8 years old male, born from non-consanguineous parents. At 7 years old, he has been diagnosed with high-risk B-ALL and CNS involvement. He received systemic and intrathecal protocol V chemotherapy. Immunology profile: hypergammaglobulinemia, normal protein response, complement, and lymphocyte populations. PCR EBV positive in blood. During leukemia treatment, he suffered from common complications.

One year later, he presented a prolonged fever with neurological involvement. EBV was found in blood and CSF, other causes where ruled out. Therefore, secondary encephalitis was assumed, although it was refractory to Rituximab. Moreover, he developed macrophage activation syndrome that remained controlled with immunosuppressive therapy. EBV copies were persistently positive and CNS showed irreversible damage.

In this context, immunology tests showed hypogammaglobulinemia and 0% B-cells after rituximab. Fluctuating hematologic parameters and decreased NKT cells. Since the unexpected outcome, WES was performed: heterozygous variation in TLR3 and NRLP12 and homozygous variation in UNC93B1 genes; all of them with uncertain significance.

Conclusions

This is a first case report with EBV chronic infection and variations in TLR3 pathway. Functional studies are mandatory to confirm the pathogenesis. It is important increase studies when evolution is unusual.

Background and Aims

Myotonic dystrophy (MD): autosomal dominant (AD) disease manifested by multisystem abnormalities including myotonia, weakness, cardiac involvement, gastrointestinal dysmotility, inverted V-shaped upper lip.

Noonan syndrome with multiple lentigines (NSML): AD, multisystem disease characterised by multiple lentigines, hypertrophic cardiomyopathy, short stature, and dysmorphic facial features. Formerly LEOPARD syndrome.

Common variable immunodeficiency (CVID): most common symptomatic primary immunodeficiency disorder in adults characterised by recurrent infections, autoimmune diseases and malignancies, low IgG and IgA, and/or IgM levels and impaired vaccine response.

Methods

Case report of clinical history and laboratory data.

Results

A 29-year old woman with family occurrence of NSML and MD1 was checked up because of her sister was treated for celiac disease (CD). She suffered from recurrent sinusitis, respiratory infections, leucopenia, thrombocytopenia, CD and AI thyroiditis, low IgG and IgA, impaired vaccine response were found. Diagnosis of CVID was set up and subcutaneous Ig replacement therapy was started. Patient´s daughter is treated with NSML and MD1, son with NSML, MD1 and extreme hypertrophic cardiomyopathy. His prognosis is unfavourable.

Genetic analyses: Pathogenic variant of PTPN11 gene, trinucleotide expansion in DMPK gene, and microduplications of 22q11 and 2q13q14.2 were observed in the family.

Conclusions

Simultaneous occurrence of NSML and MD1 is very rare. This is the first reported case of CVID with NSML and MD1. The relationship of NSML and MD1 to CVID remains undefined, further immunological evaluation in this population is necessary. Active search for hypogammaglobulinemia, especially CVID in patients with recurrent infections is warranted.

Background and Aims

C5 Deficiency is a rare autosomal recessive primary immunodeficiency disease, associated with recurrent or severe infections by Neisserial species.

We report a new mutation in a previously healthy 44-year-old woman, who developed a meningococcal sepsis by the Y strains of Neisseria who later developed sensorineural hearing loss and sensory-motor polyneuropathy responsive to steroids.

Methods

Complement study

C5 gene study

Results

C3 107 mg / dL [80-130], C4 17 mg / dL [10-22], CH50 1.1 IU / ml [35-90], MBL Activity 3.6% [10-125], Classical Activity 1.8% [ 60 - 140], Alternative Activity 2% [10 - 120]

C5 3.4 mg / dL [10.26-26.3], C6 16.8 mg / dL [7.1 - 12.8], C7 10.3 mg / dL [4-11]. C8 28.6 mg / dL[10.7-24.9].

Immunocomplexes: Negative

C5 gene study: deletion in exon 35, c.4330del, in both alleles with premature stop codon at position 1464 of the p(Glu1444Lysfs * 21). Parents were born at different locations, were not consanguineous and were heterozygous for the deletion herein described.

Conclusions

Severe meningococcal disease by Y strains is uncommon in Europe and the mutation of our patient has not been previously reported. Since parents were not consanguineous, it suggests that the carrier state for this deletion might be more frequent than expected in the spanish population. Interestingly, the clinical picture after infection, presumptively suggest an inmunomediated sensorineural hearing loss and sensory-motor polyneuropathy, although it seems not due to immunocomplexes.

Background and Aims

The complement system is a powerful tool of innate immunity to combat pathogens and maintaining host homeostasis. Pathogens activate the classical (CH50) and lectin pathway wherein the alternative pathway (AP) can serve as an “amplification loop”. The AP is also capable of autoactivation, allowing continuous monitoring for pathogens by generating small amounts of C3b. Complement factor I (CFI) plays a crucial role in the negative regulation of C3b, leading to the generation of degradation products such as C3d. Unrestricted localized overactivation of the AP due to a CFI deficiency is mainly associated with renal diseases like atypical hemolytic-uremic syndrome (aHUS) and C3 glomerulopathies (C3G).

Methods

We present two cases illustrating the laboratory hallmarks of a CFI defect.

Results

In both patients CH50, AP50 and serum complement factor C3 was reduced. A quantitative CFI deficiency was found in one patient, whereas the serum level of CFI was normal in the second patient thus initially misdiagnosed as a C3 deficiency. However, based on reduced levels of C3d, targeted sequencing revealed a compound heterozygous mutation in the CFI gene confirming a functional CFI defect. Interestingly, both patients presented with recurrent and invasive pneumococcal infections.

Conclusions

Inherited quantitative and functional deficiencies of regulator CFI can induce overactivation of the alternative pathway and typically results in reduced levels of the degradation product C3d; a diagnostic hallmark for CFI deficiency. In case of general overactivation, CFI deficiency can also present with recurrent pneumococcal infections rather than renal disease due to reduced serum C3, secondary to overconsumption.

Background and Aims

CARD protein–BCL-10–MALT1 (CBM) signalosomes are signalling complexes involved in NF KB activation. Defects in MALT1, CARD9 and CARD 11 have been described. There is only one reported case in literature of Bcl 10 deficiency. We present a child with a nonsense mutation in Bcl10 resulting in premature truncation of the protein.

Methods

Materials and method- Retrospectively analysis of a patient diagnosed as Bcl10 deficiency was analysed.

Results

6 month old boy, born of 3^rd degree consanguineous marriage with history of sibling death was referred post an intensive care admission for viral lower respiratory tract infection with palatal ulcers.A baseline immune workup showed normal lymphocyte subsets including naïve T cells and low Ig G, A and M.A differential of transient hypogammaglobulinemia of infancy was considered.On follow up, he maintained a good IgG level without replacement.

At 11 months of age, he had fever with sudden onset respiratory distress. On examination, he was tachypnoeic, maintain saturations on O2 by nasal cannula and had bilateral crepts. Evaluation showed eosinophilia and bilateral infiltrates on the chest Xray.BAL was positive for galactomannan, chest CT didn’t show any evidence of fungal infection. He was started on voriconazole with complete recovery. Next generation sequencing showed a homozygous nonsense variation in exon 2 of the BCL10 gene (chr1:85736385G>A) that resulted in a stop codon and premature truncation of the protein at codon 88. On follow up, he developed a flare of the BCG scar.

Conclusions

BCL10 and MALT 1 phenotype described include susceptibility to fungal, bacterial and viral infections as seen in our patient.

Background and Aims

Some patients with severe combined immunodeficiency may have normal T-cell counts with a severe immune-cell

activation defect in complex signaling that regulates transcriptional programs. The nuclear factor κB (NF-κB) pathway plays a role in

inflammatory and immune responses, cell adhesion, and protection against apoptosis. NF-κB pathway activation requires degradation

of the NF-κB inhibitor (IκB) proteins initiated by their phosphorylation by the IκB kinase (IKK) complex consisting of two active

kinases, IKKα and IKKβ. The IκB kinase (IKK) complex links these transcription factors to immune receptors. Several primary

immunodeficiencies in humans are caused by an impaired IKK–IκB axis. The mutation in IKBKB is characterized by

viral,fungal,bacterial infections that leads to the life-threatening consequences. The patients have

agammaglobulinemia/hypogammaglobulinemia and normal lymphocyte count.

Here, we describe an infant with a severe combined immunodeficiency with homozygous IKBKB mutation because of rarity.

Methods

The 40 day-old girl applied to the hospital with perianal and periumbilical abcesses.
She was the second child from the first generation consanguineous parents. There was history of sibling death.

Results

The investigations revealed that she had WBC:27190 mm3/L, ANC:6150 mm3/L,
ALC:10740 mm3/L, IgG:287 mg/dl, IgA:6.2 mg/dl, IgM:17.4 mg/dl, IgE:17.7 IU/ml , CD4:55.6% ,
CD8:29.7%, CD3: 83.8%, CD16+56:6.3%, CD19:7.1%, CD45:99.9%, CD11/CD18:98%. The homozygous missense
c.965_967delTCC;p.IIe322_His323delins Asn variant in IKKβ gene was identified. Although she was administrated
intensive antibiotic and immunoglobulin, she died at 5 th month of age because of septic shock.

Conclusions

The case report is first for presentation of SCID with normal ALC

agammaglobulinemia and the mutation in IKBKB gene in the literature.

Background and Aims

Autosomal recessive (AR) complete STAT1 deficiency is a rare primary immunodeficiency which causes life-threatening mycobacterial and viral infections. Prognosis of the patients are poor. Therefore, early diagnosis and appropriate therapeutic intervention are required.

Methods

Biallelic intronic STAT1 mutations were identified by whole exome sequencing (WES) and confirmed by immunoblot and flow cytometry.

Results

The patient is a 5-years-old Japanese boy who was born to nonconsanguineous parents. He received BCG vaccination at 9 months. Two months later, he developed disseminated BCG infections. Candidate gene approach for the suspicion of MSMD identified a heterozygous splice site mutation, 128+2 T>G, in STAT1 that was inherited from healthy father. Therefore, the antimycobacterial treatment was continued without confirming genetic etiology. After this episode, he repeated severe viral infections and developed vaccine induced varicella. He experienced M. malmoense positive multifocal osteomyelitis and mediastinal lymphadenitis without granuloma formation at the age of 4. Finally, an intronic mutation, 542-8A>G, in STAT1, which was inherited from healthy mother, was identified by WES at the age of 5. The STAT1 protein expression abolished in patient’s peripheral blood mononuclear cells. He is currently planned for hematopoietic stem cell transplantation.

Conclusions

The mutations identified in the current study were the first intronic mutations which caused AR complete STAT1 deficiency. Identification of intronic mutations by genomic study is sometimes challenging. Indeed, it took nearly four years to confirm molecular cause in the current study. The introduction of systemic gene expression analysis may help to improve diagnostic yield of undiagnosed patients.

Background and Aims

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in the NADPH-oxidase complex. This results in impaired respiratory oxidative species formation, a crucial step in pathogen killing. CGD is characterized by recurrent infections and granuloma formation. Five known gene defects are associated with CGD. X-linked CGD is the most prevalent and severe form. The remaining four defects cause autosomal recessive CGD of which NCF1 deficiency is the most common. We aim to provide the first report on CGD from the UAE.

Methods

We are reporting six patients from three different families, all born to consanguineous parents. Four patients are from one family. A 23-year-old male presented with recurrent skin infections and lymphadenitis complicated with skin ulcers & sinus formation. His sisters had a variable clinical presentation, ranging from hepatic abscess to recurrent lymphadenitis and failure to thrive (FTT). A 10-year-old male from the second family suffered recurrent lymphadenitis, recurrent oral thrush, pneumonia and FTT. One sister passed away at 2 years of age secondary to sepsis. A 3-year-old boy from a different family had recurrent lymphadenitis & a deep-seated scalp abscess that required incision & drainage.

Dihydrorhodamine (DHR) flow cytometeric test and next generation gene sequencing were performed.

Results

All patients had impaired neutrophil oxidative burst on DHR testing. The same homozygous, pathogenic variant c.579G>A p.(Trp193*) in the NCF1 gene was detected in affected individuals.

Conclusions

Autosomal recessive CGD is the most prevalent form of CGD in the UAE.

Background and Aims

Deletion of a group of genes in the 22q11 chromosome region is related to the DiGeorge phenotype characterized by the typical triad: hypocalcemia, conotruncal cardiac defects and thymic hypoplasia/aplasia. Whilst 0.5-1% of patients experience a complete phenotype with severe combined immunodeficiency, most of them present the partial phenotype characterized by a wide spectrum of mild immune abnormalities. The role of Nk cells in pDGS is unclear because of the few studies.

In 2015 Zheng described a reduced NK cell cytotoxic activity in a small group of 7 pDGS, reporting that this defect was caused by the deletion of crkL gene involved in the granule trafficking towards the immunological synapses. We attempted to confirm this defect in a larger group of patients with pDGS.

Methods

Twenty-eight patients with pDiGeorge syndorme were enrolled from January to June 2019. Steroid assumption in the previous three months and infections in the last month were the only exclusion criteria. A complete immunological study was performed, comprising immunoglobulins evaluation and lymphocyte proliferative test. NK cell activity test by was conducted in 19 patients evaluating the rate of effector cells and target K562 cells.

Results

NK cell activity was normal in 8 patients and elevated in 11 patients. The cytotoxic activity was strongly correlated to the NK cell number.

Conclusions

We could not confirm any defect of NK cell cytotoxic activity. All patients had at least an immunological abnormality (IgM reduction being the most common) that could self-explain the recurrence of respiratory tract infections observed in this group of patients.

Background and Aims

NEMO-deficient patients present with variable degrees of immunodeficiency. Accordingly, treatment ranges from antibiotic prophylaxis and/or IgG-substitution to allogenic hematopoietic stem cell transplantation (HSCT). The correct estimation of the immunodeficiency is essential to avoid over- as well as under-treatment. We compare the immunological phenotype of a NEMO-deficient patient with a newly-described splice site mutation that causes truncation of the NEMO zinc-finger (ZF) domain and a severe clinical course with the immunological phenotype of three NEMO-deficient patients with missense mutations and milder clinical courses and all previously published patients.

Methods

Lymphocyte subsets, proliferation and intracellular NEMO-expression were assessed by FACS. NF-κB signal transduction was determined by measuring IκBα-degradation and the production of cytokines upon stimulation with TNF-α, IL-1β and TLR-agonists in immortalized fibroblasts and whole blood respectively.

Results

The patient with truncated ZF-domain of NEMO showed low levels of IgM and IgG, reduced class-switched memory B-cells, almost complete skewing towards naïve CD45RA⁺ T-cells, impaired T-cell proliferation as well as cytokine production upon stimulation with TNF-α, IL-1β and TLR-agonists. He suffered from severe infections (sepsis, pneumonia, osteomyelitis) during infancy. In contrast, three patients with missense mutations in IKBKG presented neither skewing of T-cells towards naïvety nor impaired T-cell proliferation. They are stable on prophylactic IgG-substitution or even off any prophylactic treatment.

Conclusions

The loss of the NEMO ZF-domain and almost complete persistence of CD4⁺CD45RA⁺ naïve T-cells despite severe infections is predictive for a profound innate and adaptive immunodeficiency. Allogenic HSCT should be considered early for these patients before chronic sequelae occur.

Background and Aims

Life expectancy of CGD patients has improved but few data on its clinical course in adulthood are available. We studied long-term outcomes of uncorrected CGD adults in the UK.

Methods

Retrospective analysis of clinical records of adult CGD patients without prior corrective stem cell therapy from 8 centers across the UK.

Results

A total of 891 years of observation were collected (mean=17 years/patient) for 53 patients (mean age at symptom onset=6.2 years; 41 males: 12 females; 32:21 X-linked vs AR inheritance). 177 infectious events and 117 hospitalizations were recorded, of which skin and liver abscesses, and pulmonary infections were the most frequent. Active gastrointestinal inflammatory disease was present in 43% of adult patients, over a third of whom required major surgical intervention. CT chest scans were abnormal in 95%. Poor gas transfer was found in 62% of patients despite normal spirometry and positively correlated to bronchiectasis and inflammation on chest CT. Malignancy affected 4 patients, including 3 cases of bowel cancer. The mortality rate was 10%, predominantly related to chronic respiratory failure.

Conclusions

Survival to adulthood is frequently associated with the development of severe CGD complications and end-organ damage. Gastrointestinal and pulmonary complications were the major causes of serious morbidity and mortality in this study. These data inform counselling for pediatric and adult patients and families considering curative stem cell treatments for CGD.

Background and Aims

Many primary immunodeficiencies (PIDs) start in childhood and are life-threatening. Treatment includes haematopoietic stem cell transplant (HSCT). Due to improved techniques, more transplants are undertaken and patients are living longer. However, long-term complications can significantly affect future health and quality of life.

Previous research has focused on short-term medical outcomes and little is known about health or psychosocial outcomes in adulthood. This project is the largest and most comprehensive evaluation of long-term physical, social and psychological outcomes for adults who underwent HSCT for PID during childhood.

Methods

83 adult patients, who underwent HSCT for PID at Great Ormond Street Hospital during childhood, and at least five years previously, were recruited. Questionnaires and practical tasks assessed their current functioning and circumstances. Information was also gathered from medical notes. Data was compared with population norms and a control group of participant-nominated siblings or friends.

Results

The majority of participants reported satisfaction with their overall wellbeing. However, patients reported significantly poorer physical health-related quality of life than peers, and decreased cognitive and social functioning in some domains. These and other psychosocial, physical and cost-effectiveness outcomes will be presented.

Conclusions

Implications and suggestions for future research and service development will be discussed.

This abstract presents independent research funded by the NIHR under its Research for Patient Benefit Programme (Grant Reference Number PB-PG-0215-36145). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care.

Background and Aims

Lymphocytes are pivotal to microbial immunity, tumor surveillance and tissue homeostasis. In contrast to T, B and Natural Killer (NK) cells, the in vivo development and function of helper-like innate lymphoid cells (ILCs) remain to be fully unveiled in humans. In this study, we aimed, first, to study the kinetics of ILC recovery in adults and children following allogeneic HSCT for the curative treatment of a broad range of diseases that included tumor pathologies. Second, we aimed to assess the correlation between the reconstitution of different ILC subsets and the occurrence of GVHD.

Methods

We performed a prospective study of ILC reconstitution after HSCT in 58 patients in collaboration with the pediatric oncohematology unit of the Timone Hospital in Marseille. The retrospective study included 91 adult patients from the CRYOSTEM cohort. Quantifications of circulating T, B, NK and ILC subsets were performed by flow cytometry using BD Canto 2 and Fortessa analysers.

Results

We found that, unlike NK cells, helper-like ILCs recovered slowly, mirroring the pattern observed for T cells, with normalization at one year. The graft type and the proportion of CD34⁺cells in the graft did not significantly affect ILC reconstitution. As HSCT is often complicated by graft-versus-host disease (GVHD), we also analyzed the potential role of ILC subsets in maintaining tissue integrity in these conditions. We found that GVHD was associated with lower levels of activated and gut-homing ILC3.

Conclusions

These data support a non-redundant role of this ILC subset in lymphopenic conditions for preventing these life-threatening damages.

Background and Aims

PAD diseases have a challenging features and classification, we investigate salivary protein composition of our patients in order to reach more about the link between innate and antibody response.

Methods

We applied a top-down proteomic approach to compare, qualitatively and quantitatively, the acid soluble fraction of the salivary proteome from CVID patients and from adult healthy controls by an RP-HPLC-ESI-MS apparatus. 23 patients with predominantly antibody deficiency, 6 males and 16 females were enrolled and compared with 30 healthy controls. We focused on proteins and peptides soluble in acid solution, which were acidic proline-rich proteins (aPRPs), histatins (Hst-1, Hst-3, Hst-3 1-24, Hst-3 1-25), thymosins β4 and β10, α-defensins 1, 2, 3, and 4, statherin, P-B peptide, cystatins A, B, C, D and S, S100A7, A8, A9, and A12, such as all the known proteoforms derived by post-translational modifications (phosphorylation, oxidation, proteolytic cleavage, acetylation).

Results

Patients exhibited lower abundance of α-defensins 1-4, cystatins S1 and S2, and higher abundance of glutathionylated cystatin B and cystatin SN than controls. Patients could be clustered in two groups on the basis of different levels of SN, S1 and S2, suggesting that these proteins may play different roles in the disease

Conclusions

Quantitative variations of these pro-inflammatory and antimicrobial peptides/proteins may be related to immunodeficiency and infectious condition of the patients. The high incidence of tumors in the group with the highest level of SN, which is recognized as tumoral marker, appeared an intriguing result deserving of future investigations.

Background and Aims

Severe congenital neutropenia (SCN) is a heterogeneous group of disorders characterized by low counts of neutrophils in peripheral blood. Several genes are responsible for SCN and genotype-phenotype correlation has been established in most of them.

Methods

We reported a 2-year-old boy with chronic neutropenia who presented at the age of two weeks with anal abscesses. Immunological analysis showed normal lymphocyte subpopulations and immunoglobulins levels. Tests for autoantibodies and the neutrophil oxidative burst were normal.

Custom Next-Generation Sequencing (NGS) panel of 10 gens associated with SCN was performed.

Results

NGS revealed two monoallelic variants of uncertain clinical significance in two different candidate genes: p.Val57Met and p.Phe338Leu in JAGN1 and SLC37A4, respectively. Both variants have low allele frequency (<0.001) and in silico tools predicted a deleterious effect on protein function.

Conclusions

Due to advances in genetic testing through NGS, the knowledge about the relationship between genes and diseases and the evidence for digenic inheritance has increased in recent years. However, the genetic basis of neutropenia remains unknown in a substantial proportion of children. Here, we described a patient with chronic neutropenia and carrier of two potentially pathogenic monoallelic variants in two candidate genes. SCN is considered a monogenic disease however, a recent study suggests that an additional variant in a second causal gene could produce an impact on clinical phenotype. These data prompt us to hypothesize that the combination of these variants could produce a synergistic effect causing chronic neutropenia. Future studies are needed to explore the synergic effect of monoallelic variants in different SCN related genes.

Background and Aims

Introduction

Chronic granulomatous disease (CGD) is a primary neutrophil disease and a rare disorder associated with recurrent severe bacterial and fungal infections. Here we describe a child who admitted hospital with progressive dysphagia and diagnosed at the end with chronic granulomatous disease.

Methods

Case Report

A 10 year-old boy, was admitted to Hacettepe University Ihsan Dogramaci Children’s Hospital with 3 months history of nausea, vomiting and progressive dysphagia including both solids and liquids. In the laboratory analysis, cell blood count showed eosinophilia (700/μL), hypergamaglobulinemia and mildly high serum IgE level (127 UI/mL).Thoracic CT imaging revealed a soft tissue mass, forming increased eccentric thickness around the esophagus, starting from carina and extending till gastroesophageal junction.Transthoracic biopsy from esophagus showed eosinophilic esophagitis.

Results

The patient was tested for primary immunodeficiencies and diagnosis of CGD was confirmed by both abnormal neutrophil nitroblue tetrazolium (NBT) and dihydrorhodamine test. 1 mg/kg/day methyl prednisolon was started and given for 2 weeks for eosinophilic esophagitis. Interferon (IFN)-gamma (50mcg/m2/day twice a week) and TMP-SMX and itraconazole prophylaxis were started. Isoniazide and rifampicin were given as ppd was 15 mm.

Conclusions

Discussion

Defects in the subunits of nicotinamide dinucleotide phosphate (NADPH) oxidase enzyme complex in phagocytes cause the disease. Patients typically present with infections. CGD may cause noninfectious complications including a wide range of inflammatory diseases.In the present patient eosinophilic esophagitis causing disphagy was the first symptom of CGD. CGD cause increased incidence of GI disease. Thus, patients with distinct GI manifestations should be evaluated for CGD.

Background and Aims

Kronik mukokutanöz kandidiyazis (CMC) Candida türlerinin neden olduğu tırnakları, cildi, oral ve genital mukozayı etkileyen tekrarlayan veya kalıcı enfeksiyonlarla karakterize bulaşıcı bir fenotiptir. Th17 hücreleri, interlökin-17 (IL-17) üretir ve Candida'ya karşı konakçı mukozal bağışıklıkta önemli bir rol oynar. Son zamanlarda yapılan çalışmalar, IL-17 immünitesinin bozulmasının, CMC'nin gelişiminin altında olduğunu ortaya koydu. Burada tekrarlayan oral ve genital kandidiyazis ile başvuran bir hastada otozomal resesif bir IL17RA eksikliği sunuyoruz.

Methods

The targeted new generation sequencing PID V1 panel was used.

Results

Case report :

3-year-6-month-old male patient with consanguinity between the parents presented with thrush and genital candidiasis. It was learned that his first complaint started at 8 months of age and his complaints recurred despite receiving treatment repeatedly. Physical examination revealed oral candidiasis. Other system examinations were normal. Complete blood count, immunoglobulin and lymphocyte subgroup values were normal. The genetic analysis revealed homozygous mutation in IL17RA gene, NM­_ 014339.6 (c.787C>T (p.Arg263Ter).

Conclusions

Although basic immunological examinations are normal, patients with resistant or recurrent mucocutaneous candidiasis should be investigated for immunodeficiency.

Background and Aims

Blau syndrome (BS) is an auto-inflammatory granulomatous disease that involves abnormally increased response to IFNγ due to exaggerated NOD2 activity. Mendelian susceptibility to mycobacterial disease (MSMD) is a primary immune deficiency presenting as an infectious granulomatous disease that involves impaired production of or response to IFNγ.

Methods

NGS was performed by Illumina-based technology, functional assays included complex immune profiling was performed after stimulation by IFNγ (due to IFNGR1) and muramyl dipeptide (MDP) (in a context with NOD2 mutation). To verify the effect of the therapy, functional tests were also performed after initiation of Methotrexate treatment.

Results

We report a mother and daughter who are both heterozygous for a gain of function (GOF) NOD2 mutation previously shown to underlie BS and a dominant-negative IFNGR1 mutation previously shown to underlie MSMD. The 44-year-old mother has remained healthy, without any signs of BS and MSMD, whereas the 17-year-old daughter has displayed an altered form of BS and milder form of MSMD. Their leukocytes produced high amounts of IFNγ upon stimulation but responded poorly to this cytokine. Our findings suggest that the enhanced production of IFNγ mitigated the partial deficiency of IFNγ receptor and that the symptoms of BS were also modified. The rare association of two rare inborn errors of immunity in these kindred has thus resulted in altered forms of both disorders.

Conclusions

This experiment of nature suggests that IFNγ is an important driver of at least some manifestations of BS and that the decreased STAT1-mediated signaling via IFNγ receptor does not ameliorate the disease.