Genomic-Based Newborn Screening for Inborn Errors of Immunity: Practical and Ethical Considerations.

Inborn errors of immunity (IEI) are a group of over 450 genetically distinct conditions associated with significant morbidity and mortality, for which early diagnosis and treatment improve outcomes. Newborn screening for severe combined immunodeficiency (SCID) is currently underway in several countries, utilising a DNA-based technique to quantify T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC). This strategy will only identify those infants with an IEI associated with T and/or B cell lymphopenia. Other severe forms of IEI will not be detected. Up-front, first-tier genomic-based newborn screening has been proposed as a potential approach by which to concurrently screen infants for hundreds of monogenic diseases at birth. Given the clinical, phenotypic and genetic heterogeneity of IEI, a next-generation sequencing-based newborn screening approach would be suitable. There are, however, several ethical, legal and social issues which must be evaluated in detail prior to adopting a genomic-based newborn screening approach, and these are discussed herein in the context of IEI.

Immunoglobulin repertoire restriction characterizes the serological responses of patients with predominantly antibody deficiency.

BACKGROUND: Predominantly antibody deficiency (PAD) is the most common category of inborn errors of immunity and is underpinned by impaired generation of appropriate antibody diversity and quantity. In the clinic, responses are interrogated by assessment of vaccination responses, which is central to many PAD diagnoses. However, the composition of the generated antibody repertoire is concealed from traditional quantitative measures of serological responses. Leveraging modern mass spectrometry-based proteomics (MS-proteomics), it is possible to elaborate the molecular features of specific antibody repertoires, which may address current limitations of diagnostic vaccinology. OBJECTIVES: We sought to evaluate serum antibody responses in patients with PAD following vaccination with a neo-antigen (severe acute respiratory syndrome coronavirus-2 vaccination) using MS-proteomics. METHODS: Following severe acute respiratory syndrome coronavirus-2 vaccination, serological responses in individuals with PAD and healthy controls (HCs) were assessed by anti-S1 subunit ELISA and neutralization assays. Purified anti-S1 subunit IgG and IgM was profiled by MS-proteomics for IGHV subfamily usage and somatic hypermutation analysis. RESULTS: Twelve patients with PAD who were vaccine-responsive were recruited with 11 matched vaccinated HCs. Neutralization and end point anti-S1 titers were lower in PAD. All subjects with PAD demonstrated restricted anti-S1 IgG antibody repertoires, with usage of <5 IGHV subfamilies (median: 3; range 2-4), compared to ≥5 for the 11 HC subjects (P < .001). IGHV3-7 utilization was far less common in patients with PAD than in HCs (2 of 12 vs 10 of 11; P = .001). Amino acid substitutions due to somatic hypermutation per subfamily did not differ between groups. Anti-S1 IgM was present in 64% and 50% of HC and PAD cohorts, respectively, and did not differ significantly between HCs and patients with PAD. CONCLUSIONS: This study demonstrates the breadth of anti-S1 antibodies elicited by vaccination at the proteome level and identifies stereotypical restriction of IGHV utilization in the IgG repertoire in patients with PAD compared with HC subjects. Despite uniformly pauci-clonal antibody repertoires some patients with PAD generated potent serological responses, highlighting a possible limitation of traditional serological techniques. These findings suggest that IgG repertoire restriction is a key feature of antibody repertoires in PAD.

Can C-reactive protein be used as a surrogate marker of IL-6 in a broad array of clinical entities?

Background: C-reactive protein (CRP) is commonly performed, whereas cytokine testing is limited to research. Aims: To determine CRP correlation to cytokines IL-6, IL-1ß and TNF-α. Results: Consecutive samples (n = 307) were collected over 24 h. Ninety-six patients (31%) had acute infections, and 23 patients (7.5%) had autoimmune or inflammatory disease presentations. A strong correlation between CRP and two IL-6 assays (r = 0.74 and r = 0.71; p < 0.001) was present. CRP did not correlate with IL-1ß and TNF-α across the data set. Bacterial infection had a significantly higher CRP and IL-6 (p < 0.001), while only CRP was elevated in inflammatory and autoimmune diseases (p < 0.001). Discussion: CRP may be used as a surrogate marker of IL-6 levels in the routine diagnostic laboratories.

An appraisal of the Wilson & Jungner criteria in the context of genomic-based newborn screening for inborn errors of immunity.

Wilson and Jungner's recommendations for population-based screening have been used to guide decisions regarding candidate disease inclusion in newborn screening programs for the past 50 years. The advent of genomic-based technologies, including next-generation sequencing and its potential application to newborn screening, along with a changing landscape in terms of modern clinical practice and ethical, social, and legal considerations has led to a call for review of these criteria. Inborn errors of immunity (IEI) are a heterogeneous group of more than 450 genetically determined disorders of immunity, which are associated with significant morbidity and mortality, particularly where diagnosis and treatment are delayed. We argue that in addition to screening for severe combined immunodeficiency disease, which has already been initiated in several countries, other clinically significant IEI should be screened for at birth. Because of disease heterogeneity and identifiable genetic targets, a next-generation sequencing-based screening approach would be most suitable. A combination of worldwide experience and technological advances has improved our ability to diagnose and effectively treat patients with IEI. Considering IEI in the context of updated recommendations for population-based screening supports their potential inclusion as disease targets in newborn screening programs.

Fucosyltransferase Gene Polymorphisms and Lewisb-Negative Status Are Frequent in Swedish Newborns, With Implications for Infectious Disease Susceptibility and Personalized Medicine.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in the fucosyltransferase genes FUT2 and FUT3 have been associated with susceptibility to various infectious and inflammatory disorders. FUT variations influence the expression of human histo-blood group antigens (HBGAs) (H-type 1 and Lewis), which are highly expressed in the gut and play an important role in microbial attachment, metabolism, colonization, and shaping of the microbiome. In particular, FUT polymorphisms confer susceptibility to specific rotavirus and norovirus genotypes, which has important global health implications. METHODS: We designed a genotyping method using a nested polymerase chain reaction approach to determine the frequency of SNPs in FUT2 and FUT3, thereby inferring the prevalence of Lewisb-positive, Lewisb-negative, secretor, and nonsecretor phenotypes in 520 Swedish newborns. RESULTS: There was an increased frequency of homozygotes for the minor allele for 1 SNP in FUT2 and 4 SNPs in FUT3. Overall, 37.3% of newborns were found to have Lewis b negative phenotypes (Le (a+b-) or Le (a-b-). Using our new, sensitive genotyping method, we were able to genetically define the Le (a-b-) individuals based on their secretor status and found that the frequency of Lewis b negative newborns in our cohort was 28%. CONCLUSIONS: Given the high frequency of fucosyltransferase polymorphisms observed in our newborn cohort and the implications for disease susceptibility, FUT genotyping might play a future role in personalized health care, including recommendations for disease screening, therapy, and vaccination.

Newborn Screening for Primary Immunodeficiency Diseases: History, Current and Future Practice.

The primary objective of population-based newborn screening is the early identification of asymptomatic infants with a range of severe diseases, for which effective treatment is available and where early diagnosis and intervention prevent serious sequelae. Primary immunodeficiency diseases (PID) are a heterogeneous group of inborn errors of immunity. Severe combined immunodeficiency (SCID) is one form of PID which is uniformly fatal without early, definitive therapy, and outcomes are significantly improved if infants are diagnosed and treated within the first few months of life. Screening for SCID using T cell receptor excision circle (TREC) analysis has been introduced in many countries worldwide. The utility of additional screening with kappa recombining excision circles (KREC) has also been described, enabling identification of infants with severe forms of PID manifested by T and B cell lymphopenia. Here, we review the early origins of newborn screening and the evolution of screening methodologies. We discuss current strategies employed in newborn screening programs for PID, including TREC and TREC/KREC-based screening, and consider the potential future role of protein-based assays, targeted sequencing, and next generation sequencing (NGS) technologies, including whole genome sequencing (WGS).

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

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.