Publications by authors named "Stephan Borte"

40 Publications

Gut microbiota perturbation in IgA deficiency is influenced by IgA-autoantibody status.

Gastroenterology 2021 Mar 1. Epub 2021 Mar 1.

Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital, Huddinge, 171 77 Stockholm, Sweden. Electronic address:

Background And Aims: Immunoglobulin A (IgA) exerts its primary function at mucosal surfaces where it binds microbial antigens to regulate bacterial growth and epithelial attachment. One-third of individuals with IgA deficiency (IgAD) suffer from recurrent mucosal infections, possibly related to an altered microbiota. We here aimed to delineate the impact of IgA deficiency and the IgA-autoantibody status on the composition and functional capacity of the gut microbiota.

Methods: We performed a paired, lifestyle-balanced analysis of the effect of IgA on the gut microbiota composition and functionality based on fecal samples from IgAD subjects and IgA sufficient household members (n = 100), involving quantitative shotgun metagenomics, species-centric functional annotation of gut bacteria and strain level analyses. We supplemented the dataset with 32 IgAD subjects and examined the influence of IgA-autoantibody status on the composition and functionality of the gut microbiota.

Results: The IgAD gut microbiota exhibited decreased richness and diversity and was enriched for bacterial species encoding pathogen-related functions including multi-drug and antimicrobial peptide resistance, virulence factors, and Type III and VI secretion systems. These functional changes are largely attributed to E. coli, but were independent of E. coli strain variations and most prominent in IgAD subjects with IgA-specific autoreactive antibodies.

Conclusion: The microbiota of individuals with IgAD is enriched for species holding increased proinflammatory potential, thereby potentially decreasing the resistance to gut barrier-perturbing events. This phenotype is especially pronounced in IgAD subjects with IgA-specific autoreactive antibodies, thus warranting a screening for IgA-specific autoreactive antibodies in IgAD to identify IgAD subjects with increased risk for gastrointestinal implications.
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http://dx.doi.org/10.1053/j.gastro.2021.02.053DOI Listing
March 2021

Comparison of pre-analytical characteristics for molecular and serological diagnostics of COVID-19.

GMS Hyg Infect Control 2021 19;16:Doc03. Epub 2021 Jan 19.

Department of Laboratory Medicine, Hospital St. Georg, Leipzig, Germany.

The diagnosis of SARS-CoV-2 infection relies on RT-PCR from nasopharyngeal swabs. The pre-analytical value of different methods of material harvesting for SARS-CoV-2 are unknown. We conducted a comprehensive investigation of the pre-analytical performance for different pharyngeal sampling procedures in hospitalized patients with confirmed SARS-CoV-2 infection. In addition to swabs taken simultaneously from different locations, saliva and pharyngeal lavages were also analyzed using RT-PCR. In 10 COVID-19 patients, standard nasopharyngeal swabs detected 8 out of 10 positive patients, whereas swabs taken from the palatoglossal arch resulted in 9 correct-positive results. Brushing the posterior pharynx wall with swabs resulted in detection of 9 out of 10 positive patients with no difference using either dry swabs or liquid Amies medium. A strong correlation between Ct values of both swab materials was observed. Pharyngeal lavages yielded 6 out of 10 positive results in concordance with 85% of nasopharyngeal swabs in late-stage COVID-19 patients. Investigating 23 patients with early SARS-CoV-2 infection, pharyngeal lavages showed a concordance rate of 100% compared to nasopharyngeal swabs. The diagnostic performance of swabs taken from the palatoglossal arch in detecting SARS-CoV-2 infection is similar to that of specimens taken from the nasopharyngeal region. However, the former sampling method is associated with less discomfort and much easier to perform. Pharyngeal lavages may replace swabs for mass screening in early stages of SARS-CoV-2 infection. The predictive values are comparable, and the procedure is performed without exposing healthcare workers to transmission risks.
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http://dx.doi.org/10.3205/dgkh000374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818659PMC
January 2021

A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2.

PLoS One 2020 31;15(12):e0244824. Epub 2020 Dec 31.

Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.

SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may introduce the infection. Here we present a cheap and fast screening method based on direct RT-qPCR to detect SARS-CoV-2 in single or pooled gargle lavages ("mouthwashes"). This method detects individuals with large viral loads (Ct≤29) and we use it to test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility. This or similar approaches can be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244824PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774962PMC
January 2021

Differences of SARS-CoV-2 serological test performance between hospitalized and outpatient COVID-19 cases.

Clin Chim Acta 2020 Dec 5;511:352-359. Epub 2020 Nov 5.

Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Medical Faculty of the University and University Hospital, Leipzig, Germany. Electronic address:

Background: Serological severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays differ in the target antigen specificity, e.g. of antibodies directed against the viral spike or the nucleocapsid protein, and in the spectrum of detected immunoglobulins. The aim of the study was to evaluate the performance of two different routinely used immunoassays in hospitalized and outpatient COVID-19 cases.

Methods: The test characteristics of commercially available spike1 protein-based serological assays (Euroimmun, EI-assays), determining IgA or IgG and nucleocapsid-based assays (Virotech, VT-assays) determining IgA, IgM or IgG were compared in 139 controls and 116 hospitalized and outpatient COVID-19 cases.

Results: Hospitalized COVID-19 patients (n = 51; 115 samples) showed significantly higher concentrations of antibodies against SARS-CoV-2 and differed from outpatient cases (n = 65) by higher age, higher disease severity scores and earlier follow up blood sampling. Sensitivity of the two IgG assays was comparable in hospitalized patients tested ≥ 14 days (EI-assay: 88%, CI 67.6-99.9; VT-assay: 96%, CI 77.7-99.8). In outpatient COVID-19 cases sensitivity was significantly lower in the VT-assay (86.2%, CI 74.8-93.1) compared with the EI-assay (98.5%, CI 90.6-99.9). Assays for IgA and IgM demonstrated a lack of specificity or sensitivity.

Conclusions: Our results indicate that SARS-CoV-2 serological assays may need to be optimized to produce reliable results in outpatient COVID-19 cases who are low or even asymptomatic. Assays for IgA and IgM have limited diagnostic performance and do not prove an additional value for population-based screening approaches.
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http://dx.doi.org/10.1016/j.cca.2020.10.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642750PMC
December 2020

Comprehensive investigation of an in-hospital transmission cluster of a symptomatic SARS-CoV-2-positive physician among patients and healthcare workers in Germany.

Infect Control Hosp Epidemiol 2020 Oct 3;41(10):1209-1211. Epub 2020 Jun 3.

Department of Infectious Diseases/Tropical Medicine, Nephrology, and Rheumatology, Hospital St Georg, Leipzig, Germany.

We investigated potential transmissions of a symptomatic SARS-CoV-2-positive physician in a tertiary-care hospital who worked for 15 cumulative hours without wearing a face mask. No in-hospital transmissions occurred, despite 254 contacts among patients and healthcare workers. In conclusion, exposed hospital staff continued work, accompanied by close clinical and virologic monitoring.
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http://dx.doi.org/10.1017/ice.2020.268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298077PMC
October 2020

Immune Cellular Evaluation Following Newborn Screening For Severe T and B Cell Lymphopenia.

EJIFCC 2019 Nov 25;30(4):396-406. Epub 2019 Nov 25.

Immuno Deficiency Center Leipzig (IDCL) at Hospital St. Georg Leipzig, Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiency Diseases, Leipzig, Germany.

Newborn screening (NBS) for severe T and/or B cell lymphopenia to identify neonates with severe combined immunodeficiencies (SCID) or agammaglobulinemia rapidly after birth has paved its way into clinical practice. Debate exists on the concept and strategy for rapid verification and stratification of the cellular immune status of positively screened infants. We provide impulses for harmonization of flow cytometric approaches to allow rapid integration in the growing number of immunological laboratories involved in follow-up and subdivision of SCID and non-SCID entities.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893892PMC
November 2019

The German National Registry of Primary Immunodeficiencies (2012-2017).

Authors:
Sabine M El-Helou Anika-Kerstin Biegner Sebastian Bode Stephan R Ehl Maximilian Heeg Maria E Maccari Henrike Ritterbusch Carsten Speckmann Stephan Rusch Raphael Scheible Klaus Warnatz Faranaz Atschekzei Renata Beider Diana Ernst Stev Gerschmann Alexandra Jablonka Gudrun Mielke Reinhold E Schmidt Gesine Schürmann Georgios Sogkas Ulrich H Baumann Christian Klemann Dorothee Viemann Horst von Bernuth Renate Krüger Leif G Hanitsch Carmen M Scheibenbogen Kirsten Wittke Michael H Albert Anna Eichinger Fabian Hauck Christoph Klein Anita Rack-Hoch Franz M Sollinger Anne Avila Michael Borte Stephan Borte Maria Fasshauer Anja Hauenherm Nils Kellner Anna H Müller Anett Ülzen Peter Bader Shahrzad Bakhtiar Jae-Yun Lee Ursula Heß Ralf Schubert Sandra Wölke Stefan Zielen Sujal Ghosh Hans-Juergen Laws Jennifer Neubert Prasad T Oommen Manfred Hönig Ansgar Schulz Sandra Steinmann Klaus Schwarz Gregor Dückers Beate Lamers Vanessa Langemeyer Tim Niehues Sonu Shai Dagmar Graf Carmen Müglich Marc T Schmalzing Eva C Schwaneck Hans-Peter Tony Johannes Dirks Gabriele Haase Johannes G Liese Henner Morbach Dirk Foell Antje Hellige Helmut Wittkowski Katja Masjosthusmann Michael Mohr Linda Geberzahn Christian M Hedrich Christiane Müller Angela Rösen-Wolff Joachim Roesler Antje Zimmermann Uta Behrends Nikolaus Rieber Uwe Schauer Rupert Handgretinger Ursula Holzer Jörg Henes Lothar Kanz Christoph Boesecke Jürgen K Rockstroh Carolynne Schwarze-Zander Jan-Christian Wasmuth Dagmar Dilloo Brigitte Hülsmann Stefan Schönberger Stefan Schreiber Rainald Zeuner Tobias Ankermann Philipp von Bismarck Hans-Iko Huppertz Petra Kaiser-Labusch Johann Greil Donate Jakoby Andreas E Kulozik Markus Metzler Nora Naumann-Bartsch Bettina Sobik Norbert Graf Sabine Heine Robin Kobbe Kai Lehmberg Ingo Müller Friedrich Herrmann Gerd Horneff Ariane Klein Joachim Peitz Nadine Schmidt Stefan Bielack Ute Groß-Wieltsch Carl F Classen Jessica Klasen Peter Deutz Dirk Kamitz Lisa Lassay Klaus Tenbrock Norbert Wagner Benedikt Bernbeck Bastian Brummel Eusebia Lara-Villacanas Esther Münstermann Dominik T Schneider Nadine Tietsch Marco Westkemper Michael Weiß Christof Kramm Ingrid Kühnle Silke Kullmann Hermann Girschick Christof Specker Elisabeth Vinnemeier-Laubenthal Henriette Haenicke Claudia Schulz Lothar Schweigerer Thomas G Müller Martina Stiefel Bernd H Belohradsky Veronika Soetedjo Gerhard Kindle Bodo Grimbacher

Front Immunol 2019 19;10:1272. Epub 2019 Jul 19.

Institute for Immunodeficiency, Center for Chronic Immunodeficiency (CCI), Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs. Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel. The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1-25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0-88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE- syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%-subcutaneous; 29%-intravenous; 1%-unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy. The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment.
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http://dx.doi.org/10.3389/fimmu.2019.01272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6659583PMC
October 2020

Impaired polysaccharide responsiveness without agammaglobulinaemia in three patients with hypomorphic mutations in Bruton Tyrosine Kinase-No detection by newborn screening for primary immunodeficiencies.

Scand J Immunol 2020 Jan 30;91(1):e12811. Epub 2019 Oct 30.

Department of Pediatric Pneumology, Immunology and Intensive Care, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.

Hypomorphic mutations in the gene encoding Bruton tyrosine kinase (BTK) may result in milder phenotypes and delayed diagnosis of B-cell related immunodeficiencies due to residual BTK function. Newborn screening for kappa-deleting-recombination-excision circles (KRECs) reliably identifies classical X-linked agammaglobulinaemia (XLA) patients with profound B-cell lymphopenia at birth but has not been evaluated in patients with residual BTK function. We aimed to evaluate clinical findings, BTK function and KREC copy numbers in three patients with BTK mutations presenting with impaired polysaccharide responsiveness without agammaglobulinaemia. One patient had an invasive pneumococcal infection at the age of 4 years. All three patients (two brothers) had visible tonsils, normal to slightly decreased immunoglobulin G levels, undetectable pneumococcal antibodies despite pneumococcal conjugate vaccinations, no antibody response after a diagnostic polysaccharide vaccination as well as profound B-cell lymphopenia with residual B-cell differentiation. BTK mutations were identified by Sanger sequencing. BTK staining and phosphorylation assays were performed on peripheral B cells. KREC copy numbers were determined from dried blood spots obtained within the first week of life as well as once at the age of 8, 6 and 3 years, respectively. BTK staining showed residual protein expression. Also, residual BTK activity could be demonstrated. KREC copy numbers from dried blood spots were above the threshold set for detection of patients with profound B-cell lymphopenia. Male patients with impaired polysaccharide responsiveness should be evaluated for B-cell lymphopenia followed by BTK analyses irrespective of immunoglobulin levels or tonsil size.
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http://dx.doi.org/10.1111/sji.12811DOI Listing
January 2020

Epigenetic immune cell counting in human blood samples for immunodiagnostics.

Sci Transl Med 2018 08;10(452)

Ivana Türbachova Laboratory for Epigenetics, Epiontis GmbH, Precision for Medicine Group, 12489 Berlin, Germany.

Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3 T, CD8 T, CD4 T, and FOXP3 regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.
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http://dx.doi.org/10.1126/scitranslmed.aan3508DOI Listing
August 2018

Newborn screening using TREC/KREC assay for severe T and B cell lymphopenia in Iran.

Scand J Immunol 2018 Jun 26:e12699. Epub 2018 Jun 26.

Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran.

T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs) are recently used for detection of T or B cell lymphopenia in neonates based on region-specific cutoff levels. Here, we report cutoffs for TREC and KREC copies useful for newborn screening and/or diagnosis of primary immunodeficiency diseases (PID) in Iran. DNA was extracted from a single 3.2 mm punch of dried blood spots collected from 2160 anonymized newborns referred to two major referral health centers between 2014 and 2016. For refinement of the cutoffs, 51 patients with a definite diagnosis of severe combined immunodeficiency, X-linked agammaglobulinaemia and combined immunodeficiency, including ataxia telangiectasia, human phosphoglucomutase 3 and Janus kinase-3 deficiency, as well as 47 healthy controls were included. Samples from patients with an X-linked hyper-IgM-syndrome, Wiskott-Aldrich syndrome and DNA ligase 4 deficiency were considered as disease controls. Triplex-quantitative real-time PCR was used. Cutoffs were calculated as TRECs < 11 and KRECs < 6 copies with an ACTB > 700 copies with sensitivity of 100% for TREC and 97% for KREC. Among thirty anonymized newborn samples (1.5%) with abnormal results for TREC and/or KREC, only twenty one available cases were retested and shown to be in the normal range except for three samples (0.15%). All of the patients with a definitive diagnosis were correctly identified based on our established TREC/KREC copy numbers. Determining cutoffs for TREC/KREC is essential for correctly identifying children with PID in newborn screening. Early diagnosis of PID patients enables appropriate measures and therapies like stem cell transplantation. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1111/sji.12699DOI Listing
June 2018

Microcephaly, short stature, and limb abnormality disorder due to novel autosomal biallelic DONSON mutations in two German siblings.

Eur J Hum Genet 2018 09 14;26(9):1282-1287. Epub 2018 May 14.

Department of Pediatrics, Jena University Hospital, Jena, Germany.

Recently, variants in DONSON have been reported to cause different disorders of the microcephalic primordial dwarfism spectrum. Using whole-exome sequencing, we identified two novel, compound heterozygous DONSON variants in a pair of siblings, one of whom was previously diagnosed with Fanconi anemia. This occurred because the present cases exhibited clinical findings in addition to those of the microcephalic primordial dwarfism disorder, including severe limb malformations. These findings suggest that the DONSON and Fanconi anemia proteins could have supplementary roles in developmental processes as they have in the maintenance of genomic integrity, resulting in related disease phenotypes.
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http://dx.doi.org/10.1038/s41431-018-0128-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117362PMC
September 2018

Kappa-deleting recombination excision circle levels remain low or undetectable throughout life in patients with X-linked agammaglobulinemia.

Pediatr Allergy Immunol 2018 06 15;29(4):453-456. Epub 2018 Apr 15.

Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden.

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http://dx.doi.org/10.1111/pai.12893DOI Listing
June 2018

Study of an extended family with CTLA-4 deficiency suggests a CD28/CTLA-4 independent mechanism responsible for differences in disease manifestations and severity.

Clin Immunol 2018 03 3;188:94-102. Epub 2018 Jan 3.

Seccion de Infectología e Inmunopatología, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla, Instituto de Biomedicina de Sevilla (IBiS), Spain.

The CTLA-4 checkpoint regulates the activation of T cells. Individuals with heterozygous mutations in CTLA-4 have a complex phenotype typically characterized by antibody deficiency alongside variable autoimmunity. Despite severe disease in some individuals, others remain largely unaffected with reasons for this variation unknown. We studied a large family carrying a single point mutation in CTLA-4 leading to an amino acid change R75W and compared both unaffected with affected individuals. We measured a variety of features pertaining to T cell and CTLA-4 biology and observed that at the cellular level there was complete penetrance of CTLA-4 mutations. Accordingly, unaffected individuals were indistinguishable from those with disease in terms of level of CTLA-4 expression, percentage of Treg, upregulation of CTLA-4 upon stimulation and proliferation of CD4 T cells. We conclude that the wide variation in disease phenotype is influenced by immune variation outside of CTLA-4 biology.
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http://dx.doi.org/10.1016/j.clim.2018.01.001DOI Listing
March 2018

Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4 and CD8 T cells-clinical applications in primary immunodeficiency diagnostics.

J Allergy Clin Immunol 2017 11 7;140(5):1439-1441.e9. Epub 2017 Jun 7.

Medical Faculty, Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany.

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http://dx.doi.org/10.1016/j.jaci.2017.05.017DOI Listing
November 2017

Hematopoietic Stem Cell Transplantation in Primary Immunodeficiency Patients in the Black Sea Region of Turkey.

Turk J Haematol 2017 Dec 13;34(4):345-349. Epub 2017 Apr 13.

Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Genetic, Samsun, Turkey.

Hematopoietic stem cell transplantation is a promising curative therapy for many combined primary immunodeficiencies and phagocytic disorders. We retrospectively reviewed pediatric cases of patients diagnosed with primary immunodeficiencies and scheduled for hematopoietic stem cell transplantation. We identified 22 patients (median age, 6 months; age range, 1 month to 10 years) with various diagnoses who received hematopoietic stem cell transplantation. The patient diagnoses included severe combined immunodeficiency (n=11), Chediak-Higashi syndrome (n=2), leukocyte adhesion deficiency (n=2), MHC class 2 deficiency (n=2), chronic granulomatous syndrome (n=2), hemophagocytic lymphohistiocytosis (n=1), Wiskott-Aldrich syndrome (n=1), and Omenn syndrome (n=1). Of the 22 patients, 7 received human leukocyte antigen-matched related hematopoietic stem cell transplantation, 12 received haploidentical hematopoietic stem cell transplantation, and 2 received matched unrelated hematopoietic stem cell transplantation. The results showed that 5 patients had graft failure. Fourteen patients survived, yielding an overall survival rate of 67%. Screening newborn infants for primary immunodeficiency diseases may result in timely administration of hematopoietic stem cell transplantation.
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http://dx.doi.org/10.4274/tjh.2016.0477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774364PMC
December 2017

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

J Exp Med 2017 01 23;214(1):91-106. Epub 2016 Dec 23.

Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, SE1418 Stockholm, Sweden

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8 T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8 T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.
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http://dx.doi.org/10.1084/jem.20160849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206499PMC
January 2017

Newborn Screening for Severe Primary Immunodeficiency Diseases in Sweden-a 2-Year Pilot TREC and KREC Screening Study.

J Clin Immunol 2017 01 21;37(1):51-60. Epub 2016 Nov 21.

Department of Clinical Immunology, Karolinska University Hospital Huddinge, SE-14186, Stockholm, Sweden.

Newborn screening for severe primary immunodeficiencies (PID), characterized by T and/or B cell lymphopenia, was carried out in a pilot program in the Stockholm County, Sweden, over a 2-year period, encompassing 58,834 children. T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) were measured simultaneously using a quantitative PCR-based method on DNA extracted from dried blood spots (DBS), with beta-actin serving as a quality control for DNA quantity. Diagnostic cutoff levels enabling identification of newborns with milder and reversible T and/or B cell lymphopenia were also evaluated. Sixty-four children were recalled for follow-up due to low TREC and/or KREC levels, and three patients with immunodeficiency (Artemis-SCID, ATM, and an as yet unclassified T cell lymphopenia/hypogammaglobulinemia) were identified. Of the positive samples, 24 were associated with prematurity. Thirteen children born to mothers treated with immunosuppressive agents during pregnancy (azathioprine (n = 9), mercaptopurine (n = 1), azathioprine and tacrolimus (n = 3)) showed low KREC levels at birth, which spontaneously normalized. Twenty-nine newborns had no apparent cause identified for their abnormal results, but normalized with time. Children with trisomy 21 (n = 43) showed a lower median number of both TREC (104 vs. 174 copies/μL blood) and KREC (45 vs. 100 copies/3.2 mm blood spot), but only one, born prematurely, fell below the cutoff level. Two children diagnosed with DiGeorge syndrome were found to have low TREC levels, but these were still above the cutoff level. This is the first large-scale screening study with a simultaneous detection of both TREC and KREC, allowing identification of newborns with both T and B cell defects.
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http://dx.doi.org/10.1007/s10875-016-0347-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226987PMC
January 2017

The Extended Clinical Phenotype of 26 Patients with Chronic Mucocutaneous Candidiasis due to Gain-of-Function Mutations in STAT1.

J Clin Immunol 2016 Jan 25;36(1):73-84. Epub 2015 Nov 25.

Center for Chronic Immunodeficiency, University Medical Center Freiburg, Engesser Straße 4, 79108, Freiburg, Germany.

Purpose: Gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) result in unbalanced STAT signaling and cause immune dysregulation and immunodeficiency. The latter is often characterized by the susceptibility to recurrent Candida infections, resulting in the clinical picture of chronic mucocutaneous candidiasis (CMC). This study aims to assess the frequency of GOF STAT1 mutations in a large international cohort of CMC patients.

Methods: STAT1 was sequenced in genomic DNA from 57 CMC patients and 35 healthy family members. The functional relevance of nine different STAT1 variants was shown by flow cytometric analysis of STAT1 phosphorylation in patients' peripheral blood cells (PBMC) after stimulation with interferon (IFN)-α, IFN-γ or interleukin-27 respectively. Extended clinical data sets were collected and summarized for 26 patients.

Results: Heterozygous mutations within STAT1 were identified in 35 of 57 CMC patients (61%). Out of 39 familial cases from 11 families, 26 patients (67%) from 9 families and out of 18 sporadic cases, 9 patients (50%) were shown to have heterozygous mutations within STAT1. Thirteen distinct STAT1 mutations are reported in this paper. Eight of these mutations are known to cause CMC (p.M202V, p.A267V, p.R274W, p.R274Q, p.T385M, p.K388E, p.N397D, and p.F404Y). However, five STAT1 variants (p.F172L, p.Y287D, p.P293S, p.T385K and p.S466R) have not been reported before in CMC patients.

Conclusion: STAT1 mutations are frequently observed in patients suffering from CMC. Thus, sequence analysis of STAT1 in CMC patients is advised. Measurement of IFN- or IL-induced STAT1 phosphorylation in PBMC provides a fast and reliable diagnostic tool and should be carried out in addition to genetic testing.
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http://dx.doi.org/10.1007/s10875-015-0214-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718942PMC
January 2016

A New IL-2RG Gene Mutation in an X-linked SCID Identified through TREC/KREC Screening: a Case Report.

Iran J Allergy Asthma Immunol 2015 Aug;14(4):457-61

Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran.

Severe combined immunodeficiency (SCID) represents a rare group of primary immunodeficiency disorders (PIDs), with known or unknown genetic alterations. Here, we report a new interleukin 2 receptor, gamma chain (IL-2RG) mutation in an Iranian SCID newborn. The patient was a 6-day old boy with a family history of PID. The child was screened using a molecular-based analysis for the assessment of T cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs). Moreover, a complete immunological evaluation and gene sequencing was performed. Results showed undetectable TREC but a high level of KREC copy numbers. Flow cytometric data indicated low numbers of T and NK cells, but elevated number of B cells. A novel substitution in IL2RG: c.675 C>A, leading to p.225 Ser>Arg was found. Based on the functional analysis, the mutation is predicted to be damaging. The patient was diagnosed as a T B+ NK X-linked SCID.
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August 2015

Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville.

Pediatr Allergy Immunol 2016 Feb 23;27(1):70-7. Epub 2015 Nov 23.

Seccion de Infectología e Inmunodeficiencias, Unidad de Pediatria, Hospital Virgen del Rocío, Sevilla/Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain.

Background: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected children. T-cell-receptor-excision circles (TRECs) and kappa-deleting-recombination-excision circles (KRECs) determination from dried blood spots (DBS) identify neonates with severe T- and/or B-lymphopenia. No prospective data exist of the impact of gestational age (GA) and birth weight (BW) on TRECs and KRECs values.

Methods: TRECs and KRECs determination using triplex RT-PCR (TRECS-KRECS-β-actin-Assay) from prospectively collected DBS between 02/2014 and 02/2015 in three hospitals in Seville, Spain. Cut-off levels were TRECs < 6/punch, KRECs < 4/punch and -β-actin>700/punch. Internal (SCID, XLA, ataxia telangiectasia) and external controls (NBS quality assurance program, CDC) were included.

Results: A total of 5160 DBS were tested. Re-punch was needed in 77 samples (1.5%) due to insufficient β-actin (<700 copies/punch). Pre-term neonates (GA<37 weeks) and neonates with a BW<2500 g showed significantly lower TRECs and KRECs levels (p < 0.001). Due to repeat positive results five neonates were re-called (<0.1%): Fatal chromosomopathy (n = 1; TRECs 1/KRECs 4); extreme pre-maturity (n = 2; TRECs 0/KRECs 0 and TRECs 1/KRECs 20 copies/punch); neonates born to mothers receiving azathioprine during pregnancy (n = 2; TRECs 92/KRECs 1 and TRECs 154/KRECs 3 copies/punch). All internal and external controls were correctly identified.

Conclusions: TRECS-KRECS-β-actin-Assay correctly identifies T- and B-cell lymphopenias. Pre-maturity and low BW is associated with lower TREC and KREC levels. Extreme pre-maturity and maternal immune suppressive therapy may be a cause for false positive results of TRECs and KRECs values, respectively. To reduce the rate of insufficient samples, DBS extraction and storage need to be improved.
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http://dx.doi.org/10.1111/pai.12501DOI Listing
February 2016

SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling.

Clin Immunol 2015 Dec 14;161(2):103-9. Epub 2015 Jul 14.

Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, INSERM UMR1163, Institut IMAGINE, Paris, France; Paris Descartes University, Sorbonne Paris Cité, Imagine Institut, Paris, France. Electronic address:

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells.
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http://dx.doi.org/10.1016/j.clim.2015.07.002DOI Listing
December 2015

Novel mutations in TNFRSF7/CD27: Clinical, immunologic, and genetic characterization of human CD27 deficiency.

J Allergy Clin Immunol 2015 Sep 3;136(3):703-712.e10. Epub 2015 Apr 3.

Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden. Electronic address:

Background: The clinical and immunologic features of CD27 deficiency remain obscure because only a few patients have been identified to date.

Objective: We sought to identify novel mutations in TNFRSF7/CD27 and to provide an overview of clinical, immunologic, and laboratory phenotypes in patients with CD27 deficiency.

Methods: Review of the medical records and molecular, genetic, and flow cytometric analyses of the patients and family members were performed. Treatment outcomes of previously described patients were followed up.

Results: In addition to the previously reported homozygous mutations c.G24A/p.W8X (n = 2) and c.G158A/p.C53Y (n = 8), 4 novel mutations were identified: homozygous missense c.G287A/p.C96Y (n = 4), homozygous missense c.C232T/p.R78W (n = 1), heterozygous nonsense c.C30A/p.C10X (n = 1), and compound heterozygous c.C319T/p.R107C-c.G24A/p.W8X (n = 1). EBV-associated lymphoproliferative disease/hemophagocytic lymphohistiocytosis, Hodgkin lymphoma, uveitis, and recurrent infections were the predominant clinical features. Expression of cell-surface and soluble CD27 was significantly reduced in patients and heterozygous family members. Immunoglobulin substitution therapy was administered in 5 of the newly diagnosed cases.

Conclusion: CD27 deficiency is potentially fatal and should be excluded in all cases of severe EBV infections to minimize diagnostic delay. Flow cytometric immunophenotyping offers a reliable initial test for CD27 deficiency. Determining the precise role of CD27 in immunity against EBV might provide a framework for new therapeutic concepts.
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http://dx.doi.org/10.1016/j.jaci.2015.02.022DOI Listing
September 2015

RAC2 loss-of-function mutation in 2 siblings with characteristics of common variable immunodeficiency.

J Allergy Clin Immunol 2015 May 12;135(5):1380-4.e1-5. Epub 2014 Dec 12.

Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2014.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426222PMC
May 2015

Newborn screening for severe T and B cell lymphopenia identifies a fraction of patients with Wiskott-Aldrich syndrome.

Clin Immunol 2014 Nov 15;155(1):74-78. Epub 2014 Sep 15.

Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

The lack or marked reduction of recently formed T and B cells provides a basis for neonatal screening for severe combined immunodeficiencies (SCID) and X-linked agammaglobulinemia (XLA). Newborns with other conditions are also identified if a severe T or B cell lymphopenia is present at birth. We retrospectively analyzed Guthrie card samples from 11 children with Wiskott-Aldrich syndrome (WAS), a rare disease that requires early diagnosis and treatment, to determine whether combined T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) screening could identify these patients. 4 of 11 patients showed markedly reduced TREC or KREC copy numbers in their DBS as compared to storage-time matched controls and prospectively screened Swedish and German newborns. No correlation was observed between the WAS gene mutations, the clinical severity/course and the result of the screening assay. A diagnosis of WAS should thus be considered in newborns with positive TREC or KREC screening results.
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http://dx.doi.org/10.1016/j.clim.2014.09.003DOI Listing
November 2014

Novel NLRP12 mutations associated with intestinal amyloidosis in a patient diagnosed with common variable immunodeficiency.

Clin Immunol 2014 Oct 23;154(2):105-11. Epub 2014 Jul 23.

Ondokuz Mayıs University, Medical Faculty, Department of Pediatric Allergy and Immunology, Samsun, Turkey.

Heterozygous mutations in the NLRP12 gene have been found in patients with systemic auto-inflammatory diseases. However, the NLRP12-associated periodic fever syndromes show a wide clinical spectrum, including patients without classical diagnostic symptoms. Here, we report on a 20-year-old female patient diagnosed with common variable immunodeficiency (CVID), who developed intestinal amyloidosis and carried novel compound heterozygous mutations in NLRP12, identified by whole exome and transcriptome sequencing. CVID is a primary immunodeficiency characterized by low serum immunoglobulins, recurrent bacterial infections and development of malignancy, but it also presents with a magnitude of autoimmune features. Because of the unspecific heterogeneous clinical features of the disease, a delay in diagnosis is common. Secondary, inflammatory (AA type) amyloidosis has infrequently been observed in CVID patients. Based on our case observation and a critical review of the literature, we suggest that NLRP12 mutations might account for a small fraction of CVID patients with severe auto-inflammatory complications.
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http://dx.doi.org/10.1016/j.clim.2014.07.003DOI Listing
October 2014

Combined newborn screening for familial hemophagocytic lymphohistiocytosis and severe T- and B-cell immunodeficiencies.

J Allergy Clin Immunol 2014 Jul 29;134(1):226-8. Epub 2014 May 29.

Department of Medicine, Centre for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; Broegelmann Research Laboratory, The Gades Institute, University of Bergen, Bergen, Norway. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2014.04.026DOI Listing
July 2014

Novel diagnostic options for immunodeficiencies.

Clin Biochem 2014 Jun 20;47(9):724-5. Epub 2014 May 20.

Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Germany; Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

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http://dx.doi.org/10.1016/j.clinbiochem.2014.05.029DOI Listing
June 2014

Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots.

J Allergy Clin Immunol 2014 Jul 24;134(1):155-9. Epub 2014 Apr 24.

Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy. Electronic address:

Background: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program.

Objective: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening.

Methods: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available.

Results: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 μmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal.

Conclusion: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail.
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http://dx.doi.org/10.1016/j.jaci.2014.01.040DOI Listing
July 2014

Retrospective analysis of TREC based newborn screening results and clinical phenotypes in infants with the 22q11 deletion syndrome.

J Clin Immunol 2014 May 9;34(4):514-9. Epub 2014 Mar 9.

Department of Pediatrics, Halland Hospital Halmstad, S-301 85, Halmstad, Sweden,

Purpose: Population-based newborn screening using T-cell receptor excision circles (TREC) identifies infants with severe T-lymphopenia, seen in severe combined immunodeficiencies (SCID), but also infants with the 22q11 deletion syndrome (22q11DS). Methods for analysis of kappa-deleting recombination excision circles (KREC) help identifying infants with B-lymphopenia. We aimed to evaluate the occurrence of abnormal TREC or KREC newborn screening results in 22q11DS patients and assessed the clinical relevance of abnormal screening reports.

Methods: Simultaneous TREC and KREC analysis was performed on stored original Guthrie cards. Patients with abnormal screening reports were compared to patients with normal reports, regarding lymphocyte counts and clinical severity, obtained by retrospective analysis of medical charts.

Results: Of 48 included patients, nine (19 %) had abnormal TREC copy numbers. All 22q11DS patients with abnormal TRECs had CD3+ T-lymphopenia at the time of diagnosis, but only one patient had the complete DiGeorge syndrome. Identified 22q11DS patients with abnormal TREC copy numbers showed significantly lower CD8+ T-lymphocytes at time-of-diagnosis and were significantly more prone to viral infections, compared to 22q11DS patients with normal TREC copy numbers. All 22q11DS patients showed KREC copies within the normal range.

Conclusions: In this retrospective study a high proportion of 22q11DS patients were identified by TREC-based newborn screening. Although only one of them had the complete DiGeorge syndrome with no T-lymphocytes, all of them had T-lymphopenia and most of them had recurrent viral infections, as well as other medical problems, warranting early recognition of the syndrome.
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http://dx.doi.org/10.1007/s10875-014-0002-yDOI Listing
May 2014