Diagnostic and therapeutic approaches for primary immunodeficiencies: from newborn screening to advanced genetics and innovative therapies

Primary immunodeficiency disorders (PIDs) are rare monogenic disorders representing unique models to study the functional mechanisms of the immune system. At present, more than 300 different genes causing PIDs have been recognized. The clinical phenotype ranges between classical severe combined immunodeficiencies (SCID), with early onset and high risk of life-threatening complications during the first infancy, to combined immunodeficiencies (CID), characterized by impaired T and B cell immunity with variable phenotype spanning from recurrent and/or severe infections to autoimmunity, inflammation and malignancies. As compared to SCID, CID patients show a higher level of residual functional T cell immunity and by a later onset of the clinical manifestations and complications. Atypical manifestations of known molecular defects, variable penetrance, and interactions between genetic and environmental factors can also contribute to the phenotypic diversity of PIDs. Despite the recent progress in their genetic characterization, many patients still do not have a molecular diagnosis and their clinical management is challenging due to clinical and genetic heterogeneity of these diseases. Prompt genetic diagnosis and early specific treatment are desirable to modify the clinical course of the disease and improve the long term prognosis. Aim 1. The present project focuses on this unmet medical need aiming at the genetic characterization of PIDs of unknown origin. Innovative tools such as next generation sequencing (NGS) have been applied to perform a deep genetic analysis in a cohort of 105 patients affected by PID. A platform for the sequencing of a selected panel of genes involved in PIDs by targeted sequencing have been exploited to prioritize the analysis, before performing broader genetic analysis including whole genome or exome sequencing. In a collaborative work with the Pediatric Hospital Bambino Gesù in Rome, this study evaluated the strengths and weaknesses of targeted NGS sequencing using custom-made Ion Torrent and Haloplex panels for diagnostics and research purposes. This allowed the diagnosis of 30 (28.6%) of 105 patients tested (Cifaldi C, Brigida I, Barzaghi F et al. Front Immunol 2019, accepted manuscript). Among these, I had the chance to follow the clinical and immunological characterization of two representative cases: the first is the diagnosis of a known PID with atypical phenotype (ADA2 deficiency) and the second one is the identification of a new disease-related-gene causing a form of CID (ARPC1B deficiency). In addition, given the expanding role of newborn screening (NBS) worldwide and the importance of anticipating the diagnosis for children affected by PID to allow better outcome, I had the opportunity to participate in the setup of a protocol to extend the NBS program for PID to neonates born at the San Raffaele Hospital in Milan. Since January 2018, in 14 months of collection, upon parent’s informed consent signature, 2498 newborns (83% of the total neonates born at San Raffaele Hospital) have been screened for adenosine deaminase metabolites, (T cell receptor excision circle) TREC and (kappa-deleting recombination excision circle) KREC concentration on dried blood spots. The measurements are performed at the NEUROFABRA laboratories in Florence and at the Paediatric Hospital Meyer based on a common collaborative project sponsored by the Italian Ministry of Health. The NBS can ease not only the diagnosis of SCID but also the recognition of suspicious cases of CID far before the onset of clinical manifestations. The combined use of these approaches (NGS and NBS) can possibly reduce the elapsing time between the onset of the disease and the accomplishment of a targeted therapy. This is particularly relevant for children with SCID, such as ADA-SCID, for whom definitive therapies (hematopoietic stem cell transplantation and gene therapy) are available and ensure better outcome if performed as soon as possible. In these cases, the onset of the disease can be complicated by a concomitant infection and timely recognition of the underlying SCID is fundamental to avoid delay in the curative treatment. I had the opportunity to follow a patient affected by ADA-SCID and HCV infection. In this case, knowing the diagnosis of SCID, he was started on prophylaxis soon after birth. This probably limited the potential consequences of HCV. He was then treated with antiviral therapy and successfully underwent gene therapy. Aim 2. The study of the immunological phenotype and of the natural history of the disease are crucial in PID patients to elucidate the pathophysiological mechanisms of the disease and the clinical evolution over time. The creation of registries, containing this information represent a valuable mean to set up the most rationale therapeutic intervention. In order to follow the clinical evolution of these patients, increasingly diagnosed also thanks to NGS platforms, we set up a SCID, CID and unknown PIDs database with the support of the Italian Superior Institute of Helath. These data will possibly shed light on the response to different therapies, compare the outcomes and provide more clear indications on eligibility to hematopoietic stem cell transplantations (HSCT) vs medical therapy. Even when a precise genetic diagnosis is established, the treatment could remain challenging. Indeed, it is now evident that a single gene can present a wide range of mutations and a consequent wide range of clinical expressions of the disease, ranging from a mild immunological defect to severe immunodeficiency or immune dysregulation, with important consequences for treatment. In this light, I approached the study of two different PIDs characterized by a known immunological defect but a complete absence of genotypephenotype correlation: Activated PI3K Delta Syndrome (APDS) and Immune dysregulation Polyendocrinopathy Enteropathy X-linked (IPEX) syndrome. In these patients, due to the high variability of the clinical presentation, the therapeutic options are still debated and no general guidelines are available yet. I cooperate with the ESID-APSD registry managed by the Medical University Hospital in Freiburg. Preliminary data on the clinical features and evolution of the disease in 68 patients enrolled have been recently published. At present, the registry is following APDS patients prospectively, evaluating the effect of different therapies on the disease progression. Being aware about the importance of performing immunomonitoring in our patients, especially when a definitive guideline for treatment is lacking, we followed the immunological effects during rapamycin administration in a single APDS1 patient to ascertain the biological efficacy and correlate with the clinical data. Finally, despite the knowledge on IPEX syndrome and underlying immunological defect is continuously expanding, long-term management is still uncertain. Thus, I carried out a retrospective international cohort study on IPEX syndrome. This work allowed a preliminary data collection providing updated information on the clinical spectrum of the disease, atypical manifestations and evolution over time. In the meantime, it allowed the comparison of the outcome after HSCT vs long-term immunosuppression. The observations derived from this study resulted of interest for the patients’ management, for example the correlation between the outcome and the disease status prior HSCT as well as the increasing and beneficial use of rapamycin to control the disease. Nevertheless, this study underlined once more the need of biological markers to diagnose and follow the disease over time. To further contribute on this, I was involved in two studies on autoantibodies against cytokines and against various autoantigens (Eriksson D, J All Clin Immunol, 2019, manuscript accepted for publication). All together these studies will set the basis for patienttailored therapeutic strategies. In conclusion, the optimal management of PIDs largely depend on accurate and early diagnoses and on a profound knowledge not only of their pathogenesis but also of the disease long term evolution and their response to available therapies. Nowadays, the accessibility of next generation sequencing, newborn screening, immunological assays and registries can contribute significantly in the improvement of the management of these complex diseases supporting clinicians in the choice of timely and rationale therapeutic approaches.