Recommendations for uniform definitions used in newborn screening for severe combined immunodeficiency

BACKGROUND: Public health newborn screening (NBS) programs continuously evolve, taking advantage of international shared learning. NBS for severe combined immunodeficiency (SCID) has recently been introduced in many countries. However, comparison of screening outcomes has been hampered by use of disparate terminology and imprecise or variable case definitions for non-SCID conditions with T-cell lymphopenia. OBJECTIVES: This study sought to determine whether standardized screening terminology could overcome a Babylonian confusion and whether improved case definitions would promote international exchange of knowledge. METHODS: A systematic literature review highlighted the diverse terminology in SCID NBS programs internationally. While, as expected, individual screening strategies and tests were tailored to each program, we found uniform terminology to be lacking in definitions of disease targets, sensitivity, and specificity required for comparisons across programs. RESULTS: The study’s recommendations reflect current evidence from literature and existing guidelines coupled with opinion of experts in public health screening and immunology. Terminologies were aligned. The distinction between actionable and nonactionable T-cell lymphopenia among non-SCID cases was clarified, the former being infants with T-cell lymphopenia who could benefit from interventions such as protection from infections, antibiotic prophylaxis, and live-attenuated vaccine avoidance. CONCLUSIONS: By bringing together the previously unconnected public health screening community and clinical immunology community, these SCID NBS deliberations bridged the gaps in language and perspective between these disciplines. This study proposes that international specialists in each disorder for which NBS is performed join forces to hone their definitions and recommend uniform registration of outcomes of NBS. Standardization of terminology will promote international exchange of knowledge and optimize each phase of NBS and follow-up care, advancing health outcomes for children worldwide

Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM

<p>Abstract</p> <p>Background</p> <p>Hemophilia A represents the most common and severe inherited hemorrhagic disorder. It is caused by mutations in the F8 gene, which leads to a deficiency or dysfunctional factor VIII protein, an essential cofactor in the factor X activation complex.</p> <p>Methods</p> <p>We used long-distance polymerase chain reaction and denaturing high performance liquid chromatography for mutation scanning of the F8 gene. We designed the competitive multiplex PCR to identify the carrier with exonal deletions. In order to facilitate throughput and minimize the cost of mutation scanning, we also evaluated a new mutation scanning technique, high resolution melting analysis (HRM), as an alternative screening method.</p> <p>Results</p> <p>We presented the results of detailed screening of 122 Taiwanese families with hemophilia A and reported twenty-nine novel mutations. There was one family identified with whole exons deletion, and the carriers were successfully recognized by multiplex PCR. By HRM, the different melting curve patterns were easily identified in 25 out of 28 cases (89%) and 15 out of 15 (100%) carriers. The sensitivity was 93 % (40/43). The overall mutation detection rate of hemophilia A was 100% in this study.</p> <p>Conclusion</p> <p>We proposed a diagnostic strategy for hemophilia A genetic diagnosis. We consider HRM as a powerful screening tool that would provide us with a more cost-effective protocol for hemophilia A mutation identification.</p

The Kinetics of Early T and B Cell Immune Recovery after Bone Marrow Transplantation in RAG-2-Deficient SCID Patients

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT

The clinical utility of molecular diagnostic testing for primary immune deficiency disorders: a case based review

Primary immune deficiency disorders (PIDs) are a group of diseases associated with a genetic predisposition to recurrent infections, malignancy, autoimmunity and allergy. The molecular basis of many of these disorders has been identified in the last two decades. Most are inherited as single gene defects. Identifying the underlying genetic defect plays a critical role in patient management including diagnosis, family studies, prognostic information, prenatal diagnosis and is useful in defining new diseases. In this review we outline the clinical utility of molecular testing for these disorders using clinical cases referred to Auckland Hospital. It is written from the perspective of a laboratory offering a wide range of tests for a small developed country

Nijmegen Breakage Syndrome Detected by Newborn Screening for T Cell Receptor Excision Circles (TRECs)

PURPOSE: Severe combined immunodeficiency (SCID) encompasses a group of disorders characterized by reduced or absent T-cell number and function and identified by newborn screening utilizing T-cell receptor excision circles (TRECs). This screening has also identified infants with T lymphopenia who lack mutations in typical SCID genes. We report an infant with low TRECs and non-SCID T lymphopenia, who proved upon whole exome sequencing to have Nijmegen breakage syndrome (NBS). METHODS: Exome sequencing of DNA from the infant and his parents was performed. Genomic analysis revealed deleterious variants in the NBN gene. Confirmatory testing included Sanger sequencing and immunoblotting and radiosensitivity testing of patient lymphocytes. RESULTS: Two novel nonsense mutations in NBN were identified in genomic DNA from the family. Immunoblotting showed absence of nibrin protein. A colony survival assay demonstrated radiosensitivity comparable to patients with ataxia telangiectasia. CONCLUSIONS: Although TREC screening was developed to identify newborns with SCID, it has also identified T lymphopenic disorders that may not otherwise be diagnosed until later in life. Timely identification of an infant with T lymphopenia allowed for prompt pursuit of underlying etiology, making possible a diagnosis of NBS, genetic counseling, and early intervention to minimize complications

Relevance of laboratory testing for the diagnosis of primary immunodeficiencies: a review of case-based examples of selected immunodeficiencies

The field of primary immunodeficiencies (PIDs) is one of several in the area of clinical immunology that has not been static, but rather has shown exponential growth due to enhanced physician, scientist and patient education and awareness, leading to identification of new diseases, new molecular diagnoses of existing clinical phenotypes, broadening of the spectrum of clinical and phenotypic presentations associated with a single or related gene defects, increased bioinformatics resources, and utilization of advanced diagnostic technology and methodology for disease diagnosis and management resulting in improved outcomes and survival. There are currently over 200 PIDs with at least 170 associated genetic defects identified, with several of these being reported in recent years. The enormous clinical and immunological heterogeneity in the PIDs makes diagnosis challenging, but there is no doubt that early and accurate diagnosis facilitates prompt intervention leading to decreased morbidity and mortality. Diagnosis of PIDs often requires correlation of data obtained from clinical and radiological findings with laboratory immunological analyses and genetic testing. The field of laboratory diagnostic immunology is also rapidly burgeoning, both in terms of novel technologies and applications, and knowledge of human immunology. Over the years, the classification of PIDs has been primarily based on the immunological defect(s) ("immunophenotype") with the relatively recent addition of genotype, though there are clinical classifications as well. There can be substantial overlap in terms of the broad immunophenotype and clinical features between PIDs, and therefore, it is relevant to refine, at a cellular and molecular level, unique immunological defects that allow for a specific and accurate diagnosis. The diagnostic testing armamentarium for PID includes flow cytometry - phenotyping and functional, cellular and molecular assays, protein analysis, and mutation identification by gene sequencing. The complexity and diversity of the laboratory diagnosis of PIDs necessitates many of the above-mentioned tests being performed in highly specialized reference laboratories. Despite these restrictions, there remains an urgent need for improved standardization and optimization of phenotypic and functional flow cytometry and protein-specific assays. A key component in the interpretation of immunological assays is the comparison of patient data to that obtained in a statistically-robust manner from age and gender-matched healthy donors. This review highlights a few of the laboratory assays available for the diagnostic work-up of broad categories of PIDs, based on immunophenotyping, followed by examples of disease-specific testing