ORIGINAL RESEARCH published: 17 March 2020 doi: 10.3389/fimmu.2020.00455 Newborn Screening for Presymptomatic Diagnosis of Complement and Phagocyte Deficiencies Mahya Dezfouli 1,2 , Sofia Bergström 2 , Lillemor Skattum 3,4 , Hassan Abolhassani 1,5 , Edited by: Maja Neiman 2 , Monireh Torabi-Rahvar 6 , Clara Franco Jarava 7 , Andrea Martin-Nalda 8 , Waleed Al-Herz, Juana M. Ferrer Balaguer 9 , Charlotte A. Slade 10,11 , Anja Roos 12 , Kuwait University, Kuwait Luis M. Fernandez Pereira 13 , Margarita López-Trascasa 14 , Luis I. Gonzalez-Granado 15 , Reviewed by: Luis M. Allende-Martinez 16 , Yumi Mizuno 17 , Yusuke Yoshida 18 , Vanda Friman 19 , Hans Dieter Ochs, Åsa Lundgren 20 , Asghar Aghamohammadi 5 , Nima Rezaei 5 , Manuel Hernández-Gonzalez 7 , University of Washington School of Ulrika von Döbeln 21 , Lennart Truedsson 3 , Toshiro Hara 17 , Shigeaki Nonoyama 18 , Medicine, United States Jochen M. Schwenk 2 , Peter Nilsson 2 and Lennart Hammarström 1* Saul Oswaldo Lugo Reyes, National Institute of 1 Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska University Pediatrics, Mexico Hospital Huddinge, Stockholm, Sweden, 2 Division of Affinity Proteomics, Department of Protein Science, KTH Royal Institute *Correspondence: of Technology & SciLifeLab, Stockholm, Sweden, 3 Department of Laboratory Medicine, Section of Microbiology, Immunology Lennart Hammarström and Glycobiology, Lund University, Lund, Sweden, 4 Clinical Immunology and Transfusion Medicine, Region Skåne, Lund, lennart.hammarstrom@ki.se Sweden, 5 Research Center for Immunodeficiencies, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran, 6 Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran, 7 Immunology Department, Vall d’Hebron Research Institute, Hospital Universitari Vall d’Hebron, Specialty section: Universitat Autònoma de Barcelona, Barcelona, Spain, 8 Pediatric Infectious Diseases and Immunodeficiencies Unit, Vall This article was submitted to d’Hebron Research Institute, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain, Primary Immunodeficiencies, 9 Immunology, Hospital Universitari Son Espases/Institut d’Investigació Sanitària Illes Balears, Palma, Spain, 10 Royal a section of the journal Melbourne Hospital, Melbourne, VIC, Australia, 11 The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Frontiers in Immunology Australia, 12 Department of Microbiology and Immunology, Sint Antonius Hospital, Nieuwegein, Netherlands, 13 Department of Received: 30 November 2019 Immunology, Hospital San Pedro de Alcántara, Cáceres, Spain, 14 Departamento de Medicina, Hospital La Paz Institute for Accepted: 27 February 2020 Health Research (IdiPAZ), Universidad Autónoma de Madrid and Complement Research Group, Madrid, Spain, 15 Primary Published: 17 March 2020 Immunodeficiencies Unit, Department of Pediatrics, University Hospital 12 de Octubre, Research Institute Hospital 12 Octubre (I+12), Madrid, Spain, 16 Immunology Department, University Hospital 12 de Octubre, Research Institute Hospital 12 Citation: Octubre (I+12), Madrid, Spain, 17 Fukuoka Children’s Hospital, Kyushu University, Fukuoka, Japan, 18 Department of Dezfouli M, Bergström S, Skattum L, Pediatrics, National Defense Medical College, Saitama, Japan, 19 Department of Infectious Diseases, Institute of Biomedicine, Abolhassani H, Neiman M, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 20 Departments of Infectious Diseases, Central Torabi-Rahvar M, Franco Jarava C, Hospital, Kristianstad, Sweden, 21 Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institutet, Martin-Nalda A, Ferrer Balaguer JM, Karolinska University Hospital Solna, Stockholm, Sweden Slade CA, Roos A, Fernandez Pereira LM, López-Trascasa M, The clinical outcomes of primary immunodeficiencies (PIDs) are greatly improved by Gonzalez-Granado LI, Allende-Martinez LM, Mizuno Y, accurate diagnosis early in life. However, it is not common to consider PIDs before Yoshida Y, Friman V, Lundgren Å, the manifestation of severe clinical symptoms. Including PIDs in the nation-wide Aghamohammadi A, Rezaei N, Hernández-Gonzalez M, newborn screening programs will potentially improve survival and provide better disease von Döbeln U, Truedsson L, Hara T, management and preventive care in PID patients. This calls for the detection of Nonoyama S, Schwenk JM, Nilsson P disease biomarkers in blood and the use of dried blood spot samples, which is a part and Hammarström L (2020) Newborn Screening for Presymptomatic of routine newborn screening programs worldwide. Here, we developed a newborn Diagnosis of Complement and screening method based on multiplex protein profiling for parallel diagnosis of 22 innate Phagocyte Deficiencies. Front. Immunol. 11:455. immunodeficiencies affecting the complement system and respiratory burst function in doi: 10.3389/fimmu.2020.00455 phagocytosis. The proposed method uses a small fraction of eluted blood from dried Frontiers in Immunology | www.frontiersin.org 1 March 2020 | Volume 11 | Article 455 Dezfouli et al. Newborn Screening for Innate Primary Immunodeficiencies blood spots and is applicable for population-scale performance. The diagnosis method is validated through a retrospective screening of immunodeficient patient samples. This diagnostic approach can pave the way for an earlier, more comprehensive and accurate diagnosis of complement and phagocytic disorders, which ultimately lead to a healthy and active life for the PID patients. Keywords: primary immunodeficiency, complement deficiencies, phagocytic disorders, presymptomatic diagnosis, newborn screening, dried blood spot, protein profiling INTRODUCTION granulomatous disease (CGD) are characterized by low granulocyte counts or defects in the nicotinamide adenine Primary immunodeficiencies (PIDs) are a group of inherited dinucleotide phosphate (NADPH) oxidase pathway, leading to disorders caused by defects in different components of the conditions such as delayed wound healing, deep organ infections, immune system. PIDs may be associated with severe clinical and abscess formation. These infections may become severe outcomes if left undiagnosed or untreated (1). However, it is or even lethal, and might lead to development of malignancies uncommon to consider PIDs before the manifestation of severe or bone marrow failure (6). Early diagnosis of such disorders clinical symptoms. Recently, novel newborn screening assays allows immediate clinical intervention and prevention of severe have been explored to enable early clinical intervention and complications. Moreover, in the case of phagocytic diseases, it disease management, beginning as early as the presymptomatic might allow for early stem cell transplantation, with a future stage at birth. Newborn screening programs based on the use prospect of gene therapy. of dried blood spot (DBS) samples have revolutionized public Currently, complement functional assays, nephelometry, healthcare by detecting disorders such as phenylketonuria immunoprecipitation (IP), enzyme-linked immunosorbent assay (PKU) during the first few days of life (2). Further developments (ELISA), complete blood count and flow-cytometric cellular of this strategy introduced mass spectrometry (MS)-based characterizations are applied for diagnosis of complement and screening with improved sensitivity, specificity and capacity phagocytic disorders (5). All these methods are low-throughput, (2). Most recently, DNA-based screening methodologies hardly adaptable to population-scale performance, and can have been developed to detect severe PIDs with defects in be costly and labor-intensive to test for several disorders in adaptive immunity (2). PCR-based screening is being applied one sample. Moreover, the small quantity of blood material for diagnosing a subset of life-threatening PIDs, including in DBS significantly limits the total number of possible tests severe combined immunodeficiency (SCID) and X-linked on each sample (9). Accordingly, an alternative diagnostic agammaglobulinemia (XLA), through the measurement of method that enables multiple parallel tests and requires minimal episomal excision products of lymphocyte receptors in DBS sample volume would greatly improve the clinical procedures, samples (3). Selected countries have successfully implemented particularly in newborn screening. Considering the disease SCID-screening and several additional countries worldwide are mechanisms of complement and phagocytic defects that involve considering to include it in their national screening programs (2). the absence, or expression/structural alteration of a functional Here, we present a newborn screening based on protein protein, protein profiling of blood samples could provide a profiling that broadens the diagnosis to cover other severe fundamental diagnostic tool (10). Here, we developed a newborn forms of PIDs with innate immunity defects. The screening screening assay based on suspension bead array technology for enables parallel diagnosis of 22 disorders due to defects in parallel profiling of the main reported disease-associated proteins the complement system or phagocytic function prior to the in the complement cascade, phagocytosis and respiratory burst onset of clinical symptoms. Complement deficiencies represent function (8). The method uses a fraction of DBS corresponding 1–29% of all PID cases, (4) giving a cumulative prevalence of 1 to a sub-microliter volume of blood, and is applicable for in 20,000 live births (5), and phagocytic disorders encompass population-scale performance (Figure 1). 5–29% of PIDs, occurring at a prevalence of 1 in 250,000 live births (1, 6). However, due to the lack of comprehensive and pre-symptomatic diagnosis, the prevalence rate of these PIDs METHODS is markedly underestimated (7). Accurate and early diagnosis is vital as complement deficiencies and phagocytic disorders Blood Samples and Preparation for Protein are associated with numerous immunological complications. Profiling Complement deficiencies cause recurrent and persistent Thirty-seven anonymized healthy control samples in forms of infections in the upper respiratory tract, recurrent pneumococcal dried blood spot (DBS), serum, and whole blood (including and meningococcal infections, hereditary angioedema (HAE), 21 newborn samples) were provided by the Karolinska autoimmune complications, and renal failure due to atypical University Hospital Huddinge (Sweden) for normal range hemolytic uremic syndrome (aHUS) and glomerulonephritis identification of the targeted proteins (see Antibodies and (GN) (8). Severe congenital neutropenia (SCN) and chronic Immunoassay Procedure sections). Moreover, 41 DBS and Frontiers in Immunology | www.frontiersin.org 2 March 2020 | Volume 11 | Article 455 Dezfouli et al. Newborn Screening for Innate Primary Immunodeficiencies FIGURE 1 | Schematic view of the screening procedure. Drops of blood (∼70 µL) are dried on filter paper. Proteins are eluted and labeled from a punched disc, equal to a 4.5% fraction of one blood drop. After further dilution (1:500 dilution of blood), proteins are captured on the designed antibody-coupled bead array, and signals are analyzed with fluorescent-based readout. Stars illustrated fluorescent label. Two laser beams are applied for detection of beads (capture antibodies) and associated fluorescent signal (from labeled target proteins). matched serum samples (including five original Guthrie cards or 3 µL matched whole blood were biotinylated (referred as from the Newborn Screening Laboratory, Karolinska University protein labeling) by addition of ten times molar excess of Hospital, Sweden) with defects in one of the proteins C1, Sulfo-N-Hydroxysuccinimide-polyethyleneglycol biotin (NHS- C2, C3, C4, C5, C6, C7, C8, C9, FB, FD, FH, FI, properdin, PEG4-Biotin, ThermoFisher Scientific, USA) in 30 µL PBS- or HAX1 were obtained and prepared for retrospective T (referred as labeling solution), with incubation for 2 h at screening (Table 1). All human samples used in this study 4◦ C. Alternatively, a rapid and simultaneous protein elution were collected after informed consent and handled under and labeling was performed by submerging the DBS disc in 30 the approval of the Regional Ethical Review Board (EPN) in µL of labeling solution for 2 h, 30 min, or 10 min at ambient Stockholm, Sweden. temperature. Labeled samples were stored at −20◦ C until use. Filter papers (PerkinElmer) for DBS sampling were provided Immediately before incubation with the suspension bead array, by the Newborn Screening Laboratory at the Karolinska 1 µL of labeled samples were further diluted 50 times in University Hospital Solna (Sweden), and were prepared by PBS buffer supplemented with polyvinylpyrrolidone, polyvinyl applying 70 µL (corresponding to one drop) fresh and intact alcohol, and casein (Sigma-Aldrich). The diluted samples were whole blood onto the filter paper. Papers were dried at ambient heat-treated at 56◦ C for 30 min for signal enhancement (10) and temperature (25◦ C) for 4 h, before desiccated storage at 4◦ C were cooled down to ambient temperature for 10 min prior use. until use. DBS samples were punched in 3.2 mm in diameter The effect of heat-treatment on heat-labile complement proteins discs (corresponding to 4.5% fraction of one blood drop), and with no effect on protein profiling were previously described proteins were eluted by submerging the disc in 30 µL sterile (10). The final 1:500 dilution of eluted samples from each phosphate buffered saline supplemented with Tween 20 (PBS- disc provides enough volume for 30 tests, which corresponds T, pH 7.4, 0.1% Tween 20, Medicago-AB) for 3 days, 1 day, to a 0.1 µL volume of crude whole blood applied to each or 2 h at 4◦ C. Subsequently, proteins from the DBS eluate multiplexed measurement. Frontiers in Immunology | www.frontiersin.org 3 March 2020 | Volume 11 | Article 455 Dezfouli et al. Newborn Screening for Innate Primary Immunodeficiencies TABLE 1 | Information on the sample cohort used in retrospective screening. Diagnosed PID/disorder Number of Previous citation Provided by patient samples (if available) C1 deficiency 1# Sigma-Aldrich, product# 234401 C2 deficiency 8 (five newborns)* (10, 11) Vall d’Hebron University Hospital, Spain Son Espases University Hospital, Spain 12 Octubre Hospital, Spain Karolinska University Hospital, Sweden C3 deficiency 2# (10) Sigma-Aldrich, product# C8788 Karolinska University Hospital, Sweden C4 deficiency 1# (10) Karolinska University Hospital, Sweden C5 deficiency 1 (12) Vall d’Hebron University Hospital, Spain C6 deficiency 2 Sigma-Aldrich, product# C1288 La Paz University Hospital, Spain C7 deficiency 3 (13) Son Espases University Hospital, Spain Sahlgrenska University Hospital, Sweden National Defense Medical College, Japan C8 deficiency 1# Sigma-Aldrich, product#C1538 C9 deficiency 4 (14) Fukuoka Children’s Hospital, Japan National Defense Medical College, Japan FB deficiency 1 (15) Royal Melbourne Hospital, Australia FD deficiency 3 (16) Sint Antonius Hospital, the Netherlands FH deficiency 2# (10) Karolinska University Hospital, Sweden FI deficiency 8 (17, 18) Vall d’Hebron University Hospital, Spain Royal Melbourne Hospital, Australia San Pedro de Alcántara Hospital, Spain 12 Octubre Hospital, Spain Properdin deficiency 2 Royal Melbourne Hospital, Australia Central Hospital, Kristianstad, Sweden SCN (HAX1 deficiency) 2 (19) Tehran University of Medical Sciences, Iran Total number of patients 41 See above Healthy Controls 37 (21 newborns) Karolinska University Hospital, Sweden *Original Guthrie cards from newborn screening program in Sweden. # Available sample type is serum. Antibodies and Immunoassay Procedure An array of 42 antibodies targeting the 22 proteins and their Antibodies targeting the proteins and protein fragments C1qA, corresponding protein fragments was used for protein profiling C1qB, C1s, C2b, C3, C3a, C4A, C4B, C5, C6, C7, C8A, C8B, C9, (Table S1). FB, FD, FH, FI, Properdin, CSF3R, p22-phox, gp91-phox, p47- Fifty microliter of labeled, diluted, and heat-treated samples phox, p67-phox, p40-phox, ELANE, HAX1, human albumin and were applied to the prepared suspension bead array with bead human IgG were covalently coupled to carboxylated fluorescent- counts of minimum 30 beads per target per measurement. color-coded Luminex MagPlex microspheres (Luminex After overnight incubation in a sealed plate at ambient Corporation) according to a previously established protocol temperature on a microplate shaker (650 rpm), the beads (20). In brief, 500,000 microspheres (referred to as beads) were were washed three times in 100 µL PBS-T. The sandwich functionalized using N-Hydroxysuccinimide-polyethyleneglycol immunoassay was performed as described above, with the (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide exception of using unlabeled DBS eluate, and addition of an (EDC) chemical linkers (ThermoFisher-Scientific) according to incubation step with biotinylated secondary antibodies for 1 h the manufacturer’s protocol, and incubated with 1.65 µg of the at ambient temperature, followed by three times washes in antibody or recombinant protein in 100 µL 2-(N-Morpholino) 100 µL PBS-T (21). Subsequently, the captured biotin-labeled ethanesulfonic acid (MES) buffer at pH 5.0 (Sigma-Aldrich) proteins on beads (or detection antibodies in sandwich format) for 2 h at ambient temperature on a microplate shaker (650 were incubated with 50 µL of Streptavidin R-phycoerythrin rpm). Remaining active groups on the beads were quenched by conjugate (SAPE, ThermoFisher-Scientific) at the concentration an overnight incubation in 50 µL blocking reagent for ELISA of 5 µg/mL. After three times washes in 100 µL PBS-T and (Roche). Antibody-coupled beads were pooled in suspension to resuspension in 100 µL PBS-T, fluorescent signal intensities were be used as the suspension bead array in subsequent experiments. measured in parallel for all targets using Luminex LX200 and Frontiers in Immunology | www.frontiersin.org 4 March 2020 | Volume 11 | Article 455 Dezfouli et al. Newborn Screening for Innate Primary Immunodeficiencies FLEXMAP 3D systems (Luminex Corporation) according to the and HCLS1 associated protein X-1 (HAX1) (Table S1). The manufacturer’s instructions. deficiency level for each protein was defined based on the population of 37 healthy samples, as those with a deviation Data Analysis from healthy control values within the interquartile range of 1.5 R software version 3.2.3 and R Studio environment version (IQR ≥ 1.5). Data obtained from the retrospective screening 0.99.902 were applied for statistical analyses and data confirmed the documented disorders within the analyzed cohort visualization (22). The median fluorescence intensities (MFI) (Figure 2, Figure S2), mainly in DBS samples collected from from the beads regarding each protein target per measurement neonates with PID, compared to normal newborn samples or were used. The signal-to-background ratio was determined healthy adults (Table 1). Furthermore, the signals obtained from according to the internal negative control (data from beads with deficient samples were shown to be regained by supplementing no capture antibody). Data were normalized and fluorescent the corresponding recombinant protein into the DBS samples signals were adjusted using the “batch effect removal” function collected from the respective deficient donors (Figure S1D). from the R Limma package (23). The statistical cut-off for the The second-tier screening was also shown to be accessible in the definition of protein defects (PID level) was considered as an form of sandwich immunoassays, allowing for a more selective outlier fluorescent intensity under minimum values that deviates detection of the target proteins (Figure S3). Lastly, the screening from the 1.5 times interquartile range (IQR) under the first data were further validated by current standard clinical tests in quartile of corresponding signals for each protein measurement matched serum samples of the PID cohort (Table S2). in all healthy control samples. Values from PID patients associated with non-targeted proteins were not considered as healthy values, as no previous clinical data is available for DISCUSSION non-targeted proteins and deficiency in one complement protein can cause perturbation in the levels of other proteins due Newborn screening programs are required to meet a panel of to regulatory, compensation and feedback mechanisms. The defined criteria for the principles and practice of each diagnostic coefficient of variation (CV) was calculated for the assay as method to be considered at a public health measure. The main the ratio of the standard deviation to the mean for triplicate criteria are that the disorder needs to be a serious health issue measurements from three different samples, two different sample and late-stage treatment would cause increased morbidity and material and 43 antibodies. mortality, while early diagnosis would lead to a significantly better outcome, a treatment or effective preventive care should be RESULTS readily available for the diagnosed condition, the disorder should have a high enough incidence among the target population, the Initially, we confirmed protein elution from filter paper of costs involved should be economically balanced to adapt for three healthy controls, evaluated the protein measurements, as massive population screening, and the turnaround time from well as testing automation and reduction in turnaround time sampling to data should not exceed 48 h (2). (Figure S1A–C). The procedure was set to 30 min protein elution The genetic disorders of the innate immunity are very serious, and labeling at ambient temperature, followed by an overnight particularly when considering life-threatening infections and protein capture on an antibody-coupled bead array, and parallel angioedema. Late diagnosis would cause significantly higher fluorescent-based readout for 30 s per sample on the following morbidity leading to permanent damage to vital organs and even day. The median coefficient of variation (CV) of the method was death. A strict preventive care such as prophylactic antibody calculated to be 15. An optimal limit for blood dilution of above therapy, preventive treatment for systemic lupus erythematosus 1:800 was preferred to allow the detection of as many proteins (SLE) and HAE, as well as postponing BCG vaccination and as possible. early hematopoietic stem cell transplantation (HSCT) in CGD Subsequently, screening was performed on a sample cohort, patients would significantly reduce morbidity and mortality. including 37 healthy controls and 41 cases diagnosed with PID Currently, the cumulative prevalence of deficiencies of the innate (Table 1). The cohort included 26 newborn samples, including immunity, which could be markedly underestimated due to lack five PID cases of original Guthrie cards from the Newborn of accurate diagnosis, is within the range for newborn screening Screening Laboratory at Karolinska Hospital (Sweden) and 21 (1 in <20,000 live births in the target population). The cost per healthy DBS samples provided by the National Cord Blood assay for the proposed method is below 5 USD for the detection Biobank at Karolinska University Hospital Huddinge (Sweden). of the 22 disorders (including the costs for all reagents and Additionally, these five original Guthrie card samples from laboratory consumables) and is shown to be in the range of newborns with PID were compared with healthy DBS samples newborn screening guidelines for massive population screening. for confirmation of the newborn screening concept. The turnaround time from sampling to data is fewer than 10 h Samples were profiled for complement components C1-C9, and fits well with the time standard. complement factors FB, FD, FH, FI, and properdin, as well as A key benefit of the developed screening system is the notable the proteins related to phagocytosis and granulocyte function, number of PID-associated proteins that can be examined in including colony-stimulating factor 3 receptor (CSF3R), parallel using a limited amount of sample material. Moreover, phagocytic oxidase subunits p22-phox, gp91-phox, p47- the flexible design of the bead array content enables an optimal phox, p67-phox, and p40-phox, neutrophil elastase (ELANE) adaptation to national programs of different countries, since Frontiers in Immunology | www.frontiersin.org 5 March 2020 | Volume 11 | Article 455 Dezfouli et al. Newborn Screening for Innate Primary Immunodeficiencies FIGURE 2 | Data from retrospective PID screening. Protein profiling is done in parallel in a multiplexed measurement and obtained values are shown in separate boxplots for different disorders from 37 healthy control samples including 21 newborn dried blood spots. The deficiency level (IQR ≥ 1.5) is shown as a red baseline. Deficient samples (n = 41) are shown in colored asterisks, including five newborn dried blood spots of original Guthrie cards from newborn screening program in Sweden. SCN stands for severe congenital neutropenia. the prevalence and mechanism of PIDs are markedly different in the US was significantly adjusted from 1 in 100,000 to 1 in among ethnic groups. Nevertheless, the presented screening 58,000 live births before and after the pilot newborn screening might miss identifying cases of rare, newly discovered types of study (24). Although newborn screening of severe diseases like PIDs, or disorders caused by a dysfunctional protein expression. CGD is urgently required, the pilot screening will confirm the Updating the array to include particular antibodies that bind the usefulness of the screening for each country and will support mutated, truncated, or distorted protein fragments might further the governmental decisions on the set of other innate PIDs to identify these additional PID cases. The bead array technology include in the nation-wide screening program. Since our current can increase the sample throughput up to 384 individuals per suggested method is flexible, depending on the prevalence of instrument run, with a readout capacity of up to over 500 proteins PIDs in a specific country the cost-effectiveness assessments in in parallel (10, 20). We have shown the possibility for an extended their health-care systems can be performed to designed required array design to reach beyond the presented 22 disorders, by custom array. It is estimated that the innate immunodeficiency profiling over 1,000 protein targets in DBS samples (Figure S4). screening would benefit 20 newborns per year in Sweden and Taken together, combining the long-established routines 6,500 newborns worldwide. for DBS neonatal sampling with the inherent potential of The current study should be considered a “proof of principle” multiplexed technologies offers an appealing avenue that can be and the precursor for a pilot study with enough samples readily expanded to nationwide newborn screening for a broader size to make a decision as to cost/benefit and feasibility. range of PIDs. In the future perspective, a pilot program would Importantly, the presented approach could pave the way for address the aspects of population-scale screening prior to the an earlier, more comprehensive and accurate diagnosis of decision of implementation in the national screening programs. complement and phagocytic disorders. Correct and precise In addition, the actual prevalence rates of these PIDs will be diagnosis with an insight over several conditions could inform adjusted based on data from the pilot program. As an instance, a better understanding of these diseases by finding patients with the prevalence of severe combined immunodeficiency (SCID) specific or combination of deficiencies, in addition to showing Frontiers in Immunology | www.frontiersin.org 6 March 2020 | Volume 11 | Article 455 Dezfouli et al. Newborn Screening for Innate Primary Immunodeficiencies the differences between non-functional vs. abrogated protein YM, YY, VF, ÅL, AA, NR, AM-N, MH-G, UD, LT, TH, SN, expressions. With a more personalized diagnosis at hand, insights JS, and PN contributed with patient diagnosis and/or providing from multi-parameter assays can contribute to a more effective of reagents, materials, analysis tools. MD wrote the paper. All treatment and preventive care, which ultimately leads to a healthy authors approved the final manuscript for submission. and active life for PID patients. FUNDING DATA AVAILABILITY STATEMENT This work was supported by the Swedish Research Council The raw data supporting the conclusions of this article will be (VR) and grants provided by the Stockholm County made available by the authors, without undue reservation, to any Council (ALF). qualified researcher. ACKNOWLEDGMENTS ETHICS STATEMENT We thank all the donors and their guardians for their willingness The studies involving human participants were reviewed and to support this work. We also thank everyone at the Division approved by the Regional Ethical Review Board (EPN) in of Affinity Proteomics and the Plasma Profiling facility at Stockholm, Sweden. Written informed consent to participate in SciLifeLab. We acknowledge Dr. Jezabel Varadé, Prof. Ingrid this study was provided by the participants’ legal guardian/next Winship, Dr. Vanessa Bryant, and Dr. Heleen van Velzen-Blad of kin. for facilitating the arrangements in preparing the sample cohort. We would like to express our gratitude to Prof. Drik Roos for AUTHOR CONTRIBUTIONS support on sample collection, critical reading of the manuscript and providing helpful comments. MD, SB, LS, HA, MN, JS, PN, and LH conceived and designed the study. MD and SB planned and performed the laboratory SUPPLEMENTARY MATERIAL experiments and acquisition of data in screening. LS designed and performed the validation. MD, SB, LS, HA, MT-R, JS, PN, and The Supplementary Material for this article can be found LH analyzed and interpreted the data and revised the manuscript online at: https://www.frontiersin.org/articles/10.3389/fimmu. critically. LS, HA, CF, JF, CS, AR, LF, ML-T, LG-G, LA-M, 2020.00455/full#supplementary-material REFERENCES 10. 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Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. is permitted, provided the original author(s) and the copyright owner(s) are credited Limma powers differential expression analyses for RNA-sequencing and and that the original publication in this journal is cited, in accordance with accepted microarray studies. Nucleic Acids Res. (2015) 43:e47. doi: 10.1093/nar/ academic practice. No use, distribution or reproduction is permitted which does not gkv007 comply with these terms. Frontiers in Immunology | www.frontiersin.org 8 March 2020 | Volume 11 | Article 455