Newborn Screening for Primary Immunodeficiency Diseases

Abstract

Primary immunodeficiency diseases (PID) are a heterogeneous group of inborn errors of immunity, resulting in significant morbidity and mortality in affected infants and young children. To date, over 320 distinct genetic abnormalities resulting in PID have been described. Of interest to this thesis is severe combined immunodeficiency (SCID), where survival and longterm outcomes are significantly improved if haematopoietic stem cell transplantation (HSCT) is performed prior to 3.5 months of age. Hence, early detection of affected individuals with PID is of critical importance. The ideal infant screening strategy for PID remains controversial. Most established screening programs utilise a T cell receptor excision circle (TREC) assay to screen newborns for SCID. TREC are surrogate markers for thymic T cell output, with low levels indicative of T cell lymphopaenia. B cell lymphopaenia can also be detected through quantification of kappa-deleting recombination excision circles (KREC). This thesis presents the results of a two-year, prospective screening study for severe forms of PID manifested by T and/or B cell lymphopaenia. This was the first large-scale screening study using a multiplexed TREC/KREC assay to simultaneously detect T and B cell lymphopaenia. This assay was used to screen 58,834 newborns, resulting in the identification of three infants with PID. The findings of this study confirm the efficacy of DNA-based screening strategies to identify infants with PID and highlight the additional benefits of simultaneous screening for T and B cell lymphopaenia. TREC and KREC assays have predominantly been used in the context of newborn screening for PID, with limited application of these assays beyond the newborn period. This thesis investigated the utility of these assays in the clinical evaluation of older patients with suspected PID. The results showed that KREC levels remain low or undetectable throughout life in patients with X-linked agammaglobulinaemia (XLA). These findings, therefore, demonstrate a wider application and diagnostic utility of these assays in older children and adults. Hypogammaglobulinaemia is a feature of several forms of PID, including primary antibody deficiency disorders and combined immunodeficiencies such as SCID. Currently, there is no strategy by which to screen infants for hypogammaglobulinaemia, and measuring antibody levels in infants is confounded by the presence of maternal antibodies. This thesis presents the results of a small pilot study evaluating a transcriptomic approach to the identification of children with hypogammaglobulinaemia. Our findings show that RNA can be successfully extracted from dried bloods spots, and that immunoglobulin heavy chain gene expression assays are useful to analyse the immunoglobulin transcriptome of healthy individuals and patients with PID. In addition, this assay is able to identify children with complete deficiencies of immunoglobulin production (XLA and X-linked hyper-IgM syndrome). In approaching the era of personalised medicine, it would be advantageous not only to screen individuals for disease states, but also to identify augmentable disease susceptibility factors, thereby enabling prevention of disease. Knowledge of susceptibility factors enables the provision of patient-specific advice regarding the need for screening, therapy or prophylaxis. This thesis describes a newborn screening strategy which identifies specific SNPs in fucosyltransferase (FUT2 and FUT3) genes which determine the expression of histo-blood group antigens (HBGA) (H type 1 and Lewis). These HBGA are important for microbial attachment and metabolism and, therefore, influence an individual’s predisposition to selected infectious diseases (including rotavirus and norovirus). They also play a role in shaping the microbiome and have been associated with the development of autoimmune and inflammatory diseases. In this thesis, a genotyping method was developed to determine the frequency of SNPs in FUT2 and FUT3, and the ensuing Lewis b and secretor status. Our results demonstrated that a higher than expected proportion of newborns in the cohort were nonsecretors and Lewis b negative, predisposing them to specific infections and other diseases, with potential implications for vaccination. This thesis contributes to the literature regarding newborn screening strategies for PID, providing evidence for the efficacy of combined TREC/KREC screening to identify infants with severe forms of PID manifested by T and/or B cell lymphopaenia. It outlines the utility of screening tests beyond the newborn period as tools for the diagnostic work-up of patients with PID. Evidence is provided for the utility of a newborn screening approach for SNPs conferring disease susceptibility, which has implications for the provision of personalised, precision medicine. These findings are discussed and reviewed with respect to the current international status of newborn screening for PID, highlighting differences in screening approaches. This thesis reviews application of other methods, including protein-based assays and targeted sequencing to expand the range of currently screened diseases. The potential utility of screening newborns using up-front next generation sequencing (whole exome and whole genome sequencing) is discussed, highlighting the key considerations prior to adopting this approach