Newborn screening for severe primary immunodeficiencies

Primary immunodeficiencies (PID) are congenital disorders of immune competence, which are mainly characterized by a pathological susceptibility to infection. This is often accompanied by severe recurrent infections with drug-resistant, long progressions. In addition, there are associated immune regulation disorders, which may manifest themselves in granuloma formation, autoimmunity, recurrent fever, eczema, lymphoproliferation and chronic intestinal inflammation. More than 240 disease entities have been defined so far as PID, and just as extensive is the spectrum of clinical severity. While the most common congenital immunodeficiencies, such as selective IgA deficiency or C2 complement deficiency, have a mild phenotype which often remains undetected, severe PID are characterized by significant mortality in the first years of life, as well as a serious morbidity with irreversible organ damage. This applies in particular to PID that are defined by the absence or functional anergy of T-lymphocytes (severe combined immunodeficiency; SCID) or B-lymphocytes (e.g. X-linked agammaglobulinemia; XLA). Patients with such severe congenital immunodeficiencies appear to be in perfect health at birth, yet show initial manifestations of SCID between the 14th day and the 4th month following birth; in patients with XLA usually between the 8th and 16th month after birth. Albeit increasingly becoming appreciated as a relevant health problem, there is a lack of diagnostic procedures and screening profiles that would allow earliest possible diagnosis of patients with severe PID on a population scale. As a superior prognosis could be given upon prompt diagnosis and immediate adequate treatment, one strategy to improve the outcome of severe PID shall be to test newborns for the presence of T and B cells. With the aim to develop a simple and reliable test for newborn screening using the established dried blood sampling system (Guthrie cards), a multiplex real-time quantitative qPCR assay for the quantitation of T cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) as surrogate markers of T and B cell development was designed and evaluated (publication I). This assay was further extended to allow detection of newborns with an inversion of the UNC13D gene, causing a severe PID characterized as familial hemophagocytic lymphohistiocytosis (publication III). Furthermore, the feasibility to identify several other severe PID, characterized by a functional defect of T- and B-cell interation (combined immunodeficiency diseases), was assessed using IgA-protein detection in neonatal Guthrie cards. However, this assay provided evidence of a maternal transfer mechanism for IgA, thereby preventing the use of this assay as a screening tool for severe PID (publication II). Finally, the TREC-KREC newborn screening assay was further improved in terms of assay performance and evaluated in retrospective cohorts of patients with the DiGeorge syndrome, or Down syndrome (publications IV and V). In addition, novel second-tier assays for comfirmation of the 22q11 microdeletion or the chromosome 21 triplication have been designed and succesfully tested with neonatal samples. In summary, new assays and concepts for newborn screening of severe primary immunodeficeincies were designed and benchmarked in retrospective and prospective neonatal dried blood spot samples, thereby underlining the potential of this preventive health care strategy

Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual

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

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 approach to detect SARSCoV-2 in single or pooled gargle lavages (“mouthwashes”). With this approach, we 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 could be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics

Long-Term Follow-Up of Newborns with 22q11 Deletion Syndrome and Low TRECs.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadBackground: Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). Purpose: To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. Methods: Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. Results: At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. Conclusions: This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. Clinical implications: This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices. Keywords: 22q11.2 deletion syndrome; DiGeorge syndrome; T lymphopenia; TREC; long-term outcome; newborn screening; severe combined immunodeficiency.University of Gothenburg Regional research grant Region Halland Swedish Research Council European Commission Queen Silvia Jubilee Foundation Swedish Primary Immunodeficiency Organization Sparbanken Foundation Varberg Frimurare Barnhusdirektionen Foundation Gothenburg Medical Society Medical Faculty at Umea University Cancer Research Foundation in Northern Sweden Swedish government county councils, the ALF-agreement Umea University Vasterbottens County Counci

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

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

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

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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

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

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

Migraine polygenic risk score associates with efficacy of migraine-specific drugs

Objective To assess whether the polygenic risk score (PRS) for migraine is associated with acute and/or prophylactic migraine treatment response. Methods We interviewed 2,219 unrelated patients at the Danish Headache Center using a semistructured interview to diagnose migraine and assess acute and prophylactic drug response. All patients were genotyped. A PRS was calculated with the linkage disequilibrium pred algorithm using summary statistics from the most recent migraine genome-wide association study comprising ∼375,000 cases and controls. The PRS was scaled to a unit corresponding to a twofold increase in migraine risk, using 929 unrelated Danish controls as reference. The association of the PRS with treatment response was assessed by logistic regression, and the predictive power of the model by area under the curve using a case-control design with treatment response as outcome. Results A twofold increase in migraine risk associates with positive response to migraine-specific acute treatment (odds ratio [OR] = 1.25 [95% confidence interval (CI) = 1.05–1.49]). The association between migraine risk and migraine-specific acute treatment was replicated in an independent cohort consisting of 5,616 triptan users with prescription history (OR = 3.20 [95% CI = 1.26–8.14]). No association was found for acute treatment with non–migraine-specific weak analgesics and prophylactic treatment response. Conclusions The migraine PRS can significantly identify subgroups of patients with a higher-than-average likelihood of a positive response to triptans, which provides a first step toward genetics-based precision medicine in migraine

Newborn Screening for Primary Immunodeficiencies: Focus on Severe Combined Immunodeficiency (SCID) and Other Severe T-Cell Lymphopenias

Primary immunodeficiencies (PID) are congenital disorders of immune competence, which are mainly characterized by a pathological susceptibility to infection. More than 240 PID disease entities have been defined so far, accounting for a broad spectrum of clinical symptoms and severity. Severe PID are increasingly becoming appreciated as a relevant health problem, and diagnostic procedures and screening profiles to allow earliest possible diagnosis on a population scale have already been developed in the USA and few European countries. The most severe PID are characterized by significant mortality in the first years of life, as well as serious morbidity with irreversible organ damage. This applies in particular to PID that are defined by the absence or functional anergy of T-lymphocytes (severe combined immunodeficiency; SCID) or B-lymphocytes (e.g., X-linked agammaglobulinemia; XLA). A strategy to improve the outcome of severe PID by prompt diagnosis and immediate adequate treatment is screening newborns for the presence of T and B cells