Search Results (16)

Background/Objectives: Genetic newborn screening (NBS) has already entered the phase of common practice in many countries. In Germany, spinal muscular atrophy (SMA), severe combined immunodeficiency (SCID) and sickle cell disease (SCD) are currently a mandatory part of NBS. Here, we describe the experience of six years of genetic NBS including the prevalence of those three diseases in Germany. Methods: Samples and nucleic acids were extracted from dried blood spot cards, commonly used for NBS. A qPCR assay was used to detect disease-causing variants for SMA and SCD, and the detection of T-cell receptor excision circles (TRECs) was performed for SCID screening. Results: The results of the NBS of over 1 million newborns for SMA, approximately 770,000 for SCID and over 410,000 for SCD are discussed in detail. In these newborns, we have identified 121 cases of SMA, 15 cases of SCID and syndrome-based immunodeficiencies and 77 cases of SCD or β-thalassemia. Conclusions: The flexibility of multiplex qPCR is assessed as an effective tool for incorporating different molecular genetic markers for screening. The processing of dried blood spot (DBS) filter cards for molecular genetic assays and the assays are described in detail; turn-around times and cost estimations are included to give an insight into the processes and discuss further options for optimization. The identified cases are in the range expected for the total number of screened newborns, but present a more exact view on the actual prevalences for Germany. Full article

The expanded newborn screening (NBS) program in the Russian Federation was initiated in 2023, among which severe combined immunodeficiency (SCID) is screened using TREC/KREC assays. Here, we report a rare case of a TP63-associated disease identified through this NBS program. Dried blood spots from newborns were initially screened for TREC/KREC levels, and those with values below the cut-off underwent confirmatory testing and further genetic analysis, including whole-exome sequencing (WES). A male newborn was identified with significantly reduced TREC values, indicative of T cell lymphopenia. Genetic analysis revealed a heterozygous NM_003722.5:c.1027C>T variant in TP63, leading to the p.(Arg343Trp) substitution within the DNA binding domain. This mutation has been previously associated with Ectrodactyly–Ectodermal Dysplasia–Cleft lip/palate syndrome (EEC) syndrome and shown to reduce the transactivation activity of TP63 in a dominant-negative manner. This case represents one of the few instances of immune system involvement in a patient with a TP63 mutation, highlighting the need for further investigation into the immunological aspects of TP63-associated disorders. Our findings suggest that comprehensive immunological evaluation should be considered for patients with TP63 mutations to better understand and manage potential immune dysfunctions. Full article

Pneumonia is an acute infectious disease with high morbidity and mortality rates. Pneumonia’s development, severity and outcome depend on age, comorbidities and the host immune response. In this study, we combined theoretical and experimental investigations to characterize pneumonia and its comorbidities as well as to assess the host immune response measured by TREC/KREC levels in patients with pneumonia. The theoretical study was carried out using the Columbia Open Health Data (COHD) resource, which provides access to clinical concept prevalence and co-occurrence from electronic health records. The experimental study included TREC/KREC assays in young adults (18–40 years) with community-acquired (CAP) (n = 164) or nosocomial (NP) (n = 99) pneumonia and healthy controls (n = 170). Co-occurring rates between pneumonia, sepsis, acute respiratory distress syndrome (ARDS) and some other related conditions common in intensive care units were the top among 4170, 3382 and 963 comorbidities in pneumonia, sepsis and ARDS, respectively. CAP patients had higher TREC levels, while NP patients had lower TREC/KREC levels compared to controls. Low TREC and KREC levels were predictive for the development of NP, ARDS, sepsis and lethal outcome (AUC_TREC in the range 0.71–0.82, AUC_KREC in the range 0.67–0.74). TREC/KREC analysis can be considered as a potential prognostic test in patients with pneumonia. Full article

Severe combined immunodeficiency is a rare inherited disorder, which, if untreated, invariably proves fatal in late infancy or early childhood. With treatment, the prognosis is much improved. Early treatment of the siblings of cases, before they become symptomatic, has shown considerable improvements in outcomes. Based on this and the development of a test that can be used on the whole population of neonates (measurement of T-cell receptor excision circles—TRECs), many countries have added it to their routine newborn bloodspot screening programmes. The UK National Screening Committee (UKNSC) has considered whether SCID should be added to the UK screening programme and concluded that it was likely to be cost effective, but that there were a number of uncertainties that should be resolved before a national roll-out could be recommended. These include some aspects of the test, such as: cost; the use of different assays and cut-off levels to reduce false positive rates, while maintaining sensitivity; the overall benefits of screening for disease outcome in patients with SCID and other identified disorders; the need for a separate pathway for premature babies; the acceptability of the screening programme to parents of babies who have normal and abnormal (both true and false positive) screening results. To achieve this, screening of two thirds of babies born in England over a two-year period has been planned, beginning in September 2021. The outcomes and costs of care of babies identified by the screening will be compared with those of babies identified with SCID in the rest of the UK. The effect of the screening programme on parents will form part of a separate research project. Full article

An evaluation program for newborn screening for Severe Combined Immunodeficiency began in England in September 2021 based on TREC analysis. Flow cytometry is being used as the follow up diagnostic test for patients with low/absent TRECS. The immunology laboratories have established a protocol and values for diagnosing SCID, other T lymphopenias and identifying healthy babies. This commentary describes the flow cytometry approach used in England to define SCID, T lymphopenia and normal infants after a low TREC result. It provides background to the flow cytometry assays being used and discusses the need to monitor and potentially change these values over time. Full article

Newborn screening (NBS) programs continue to expand due to innovations in both test methods and treatment options. Since the introduction of the T-cell receptor excision circle (TREC) assay 15 years ago, many countries have adopted screening for severe combined immunodeficiency (SCID) in their NBS program. SCID became the first inborn error of immunity (IEI) in population-based screening and at the same time the TREC assay became the first high-throughput DNA-based test in NBS laboratories. In addition to SCID, there are many other IEI that could benefit from early diagnosis and intervention by preventing severe infections, immune dysregulation, and autoimmunity, if a suitable NBS test was available. Advances in technologies such as KREC analysis, epigenetic immune cell counting, protein profiling, and genomic techniques such as next-generation sequencing (NGS) and whole-genome sequencing (WGS) could allow early detection of various IEI shortly after birth. In the next years, the role of these technical advances as well as ethical, social, and legal implications, logistics and cost will have to be carefully examined before different IEI can be considered as suitable candidates for inclusion in NBS programs. Full article

All newborn screening programs screen for severe combined immunodeficiency by measurement of T-cell receptor excision circles (TRECs). Herein, we report our experience of reporting TREC assay results as multiple of the median (MoM) rather than using conventional copy numbers. This modification simplifies the assay by eliminating the need for standards with known TREC copy numbers. Furthermore, since MoM is a measure of how far an individual test result deviates from the median, it allows normalization of TREC assay data from different laboratories, so that individual test results can be compared regardless of the particular method, assay, or reagents used. Full article

After it was demonstrated in 2005 that T cell receptor excision circle (TREC) quantification for dried blood spot (DBS) samples on Guthrie cards is an effective means of SCID screening and following several pilot studies, the practice was formally recommended in the US in 2010. More and more countries have adopted it since then. In France, before the health authorities could recommend adding SCID to the list of five diseases that were routinely screened for, feasibility and cost-effectiveness studies had to be conducted with a sufficiently large cohort of neonates. We carried out three such studies: The first sought to verify the effectiveness of the assay. The second, DEPISTREC, evaluated the feasibility of universal SCID screening in France and assessed the clinical benefit and economic advantage it would provide. Through the third study, NeoSKID, still under way and to continue until recommendations are issued, we have been offering SCID screening in the Pays de la Loire region of France. This review briefly describes routine newborn screening (NBS) and management of primary immunodeficiency diseases (PIDs) in France, and then considers the lessons from our studies and the status of SCID screening implementation within the country. Full article

Numerous studies have shown evidence supporting the benefits of universal newborn screening for primary immunodeficiencies (PID) and for Spinal Muscular Atrophy (SMA). We have developed a four-plex, real-time PCR assay to screen for Severe Combined Immune Deficiencies (SCID), X-linked agammaglobulinemia (XLA), and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS). A simple, high-throughput, semi-automated DNA extraction method was developed for a Janus liquid handler that can process 384 DBS punches in four 96-well plates in just over one hour with sample tracking capability. The PCR assay identifies the absence of exon 7 in the SMN1 gene, while simultaneously evaluating the copy number of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules. Additionally, the amplification of a reference gene, RPP30, was included in the assay as a quality/quantity indicator of DNA isolated from the DBS. The assay performance was demonstrated on over 3000 DNA samples isolated from punches of putative normal newborn DBS. The reliability and analytical accuracy were further evaluated using DBS controls, and contrived and confirmed positive samples. The results from this study demonstrate the potential of future molecular DBS assays, and highlight how a multiplex assay could benefit newborn screening programs. Full article

The implementation of newborn screening for severe combined immunodeficiency (SCID) in the Netherlands is a multifaceted process in which several parties are involved. The Dutch Ministry of Health adopted the advice of the Dutch Health Council to include SCID in the Dutch newborn screening program in 2015. As newborn screening for SCID is executed with a new, relatively expensive assay for the Dutch screening laboratory, an implementation pilot study is deemed instrumental for successful implementation. A feasibility study was performed in which the practicalities and preconditions of expanding the newborn screening program were defined. Cost-effectiveness analysis (CEA) indicated that SCID screening in the Netherlands might be cost-effective, recognizing that there are still many uncertainties in the variables underlying the CEA. Data and experience of the pilot study should provide better estimates of these parameters, thus enabling the actualization of CEA results. Prior to the implementation pilot study, a comparison study of two commercially available SCID screening assays was performed. A prospective implementation pilot study or so-called SONNET study (SCID screening research in the Netherlands with TRECs) started in April 2018 and allows the screening for SCID of all newborns in three provinces of the Netherlands for one year. Based on the results of the SONNET study, the Dutch Ministry of Health will make a final decision about national implementation of newborn screening for SCID in the Netherlands. Full article

The incidence of Severe Combined Immunodeficiency (SCID) in Manitoba, (1/15,000), is at least three to four times higher than the national average and that reported from other jurisdictions. It is overrepresented in two population groups: Mennonites (ZAP70 founder mutation) and First Nations of Northern Cree ancestry (IKBKB founder mutation). We have previously demonstrated that in these two populations the most widely utilized T-cell receptor excision circle (TREC) assay is an ineffective newborn screening test to detect SCID as these patients have normal numbers of mature T-cells. We have developed a semi-automated, closed tube, high resolution DNA melting procedure to simultaneously genotype both of these mutations from the same newborn blood spot DNA extract used for the TREC assay. Parallel analysis of all newborn screening specimens utilizing both TREC analysis and the high-resolution DNA procedure should provide as complete ascertainment as possible of SCID in the Manitoba population. Full article

Background: Quantification of T-cell-receptor-excision circles (TRECs) and kappa-deleting-recombination-excision circles (KRECs) from dried blood spots (DBS) allows detection of neonates with severe T-cell and/or B-cell lymphopenia that are potentially affected by severe combined immunodeficiency (SCID), as well as X-linked agammaglobulinemia (XLA). Methods: Determination of TRECs and KRECs using a triplex RT-PCR (TRECS-KRECS-β-actin) assay from prospectively collected DBS between February 2014 and December 2016 in three hospitals in Seville, Spain. Cut-off levels were TRECs < 6/punch, KRECs < 4/punch and b-actin > 700/punch. Internal (SCID, XLA, ataxia telangiectasia) and external controls (CDC) were included. Results: A total of 8943 DBS samples obtained from 8814 neonates were analysed. Re-punching was necessary in 124 samples (1.4%) due to insufficient β-actin values (<700 copies/punch). Preterm neonates (GA < 37 weeks) and neonates with a BW < 2500 g showed significantly lower TRECs and KRECs levels (p < 0.001). Due to repeated pathological results, ten neonates were re-sampled (0.11%), of which five neonates (0.055%) confirmed the pathological results: one case was a fatal chromosomopathy (TRECs 1/KRECs 4); two were extreme premature newborns (TRECs 0/KRECs 0 and TRECs 1/KRECs 20 copies/punch); and 2 neonates were born to mothers receiving azathioprine during pregnancy (TRECs 92/KRECs 1 and TRECs 154/KRECs 3 copies/punch). All controls were correctly identified. Conclusions: Severe T- and B-cell lymphopenias were correctly identified by the TRECS-KRECS-β-actin assay. Prematurity and low BW are associated with lower TREC and KREC levels. Extreme prematurity and maternal immune suppressive therapy can cause false positive results of TRECs and KRECs values. Full article

Primary immunodeficiency diseases (PID) are a heterogeneous group of disorders caused by inborn errors of immunity, with affected children presenting with severe, recurrent or unusual infections. Over 300 distinct genetic molecular abnormalities resulting in PID have been identified, and this number continues to rise. Newborn screening for PID has been established in many countries, with the majority of centers using a PCR-based T cell receptor excision circle (TREC) assay to screen for severe combined immunodeficiency (SCID) and other forms of T cell lymphopenia. Multiplexed screening including quantitation of kappa-recombining exclusion circles (KREC) has also been described, offering advantages over TREC screening alone. Screening technologies are also expanding to include protein-based assays to identify complement deficiencies and granulocyte disorders. Given the rapid advances in genomic medicine, a potential future direction is the application of next-generation sequencing (NGS) technologies to screen infants for a panel of genetic mutations, which would enable identification of a wide range of diseases. However, several ethical and economic issues must be considered before moving towards this screening strategy. Full article

In the US, the assay of T cell receptor excision circles (TRECs) in newborn dried blood spot specimens to detect severe combined immunodeficiency (SCID) was first piloted in 2008 in the state of Wisconsin. It has been rapidly adopted with 49 states and Puerto Rico now either routinely screening all newborns or planning to do so in 2017. Advances in SCID NBS over the last 9 years have revolutionized the ability to detect SCID and has led to profound improvement in outcomes of affected children. Full article

Infants born with T cell lymphopenias, especially severe combined immunodeficiency (SCID) are at risk for serious, often fatal infections without intervention within the first year or two of life. The majority of these disorders can be detected through the use of the T cell recombination excision circle assay (TREC assay.) The TREC assay detects the presence of non-replicating, episomal DNA that is formed during T cell development. This assay initially developed to measure thymic output during aging and HIV infection, has undergone modifications for the purpose of newborn screening (NBS) for SCID. To meet the requirements for inclusion on NBS panels, the assay needed to utilize blood from dried blood spots on NBS cards, and be both sensitive and specific, avoiding the costs of false positives. Currently, the assay relies upon real time, quantitative PCR (RT-qPCR) to detect TRECs in punches taken from dried blood spots. This review seeks to highlight some of the early work leading up to the initial implementation of the TREC assay for SCID detection, and the subsequent revisions made to optimize the assay. Full article