Abnormal T-cell Receptor Excision Circle Research Articles

Increased mortality in infants with abnormal T-cell receptor excision circles.

T-Cell Receptor Excision Circles based newborn screening (TREC-NBS) allows for early detection of T-cell lymphopenia in infants with primary immunodeficiency disorders (PIDD). The utility of abnormal TREC-NBS in infants without PIDD is not well studied. We sought to evaluate the association of abnormal TREC-NBS with mortality. 365,207 TREC-NBS from October 2011 to December 2014 were reviewed. 467 newborns had abnormal screens and did not meet the criteria for a PIDD diagnosis. Cases were matched to controls (1:3) based on gestational age, birth weight, neonatal intensive care unit status (NICU), and race. Data were obtained through NBS, birth and death certificates records from the Michigan Department of Health and Human Services (MDHHS) databases. Infants with abnormal TREC-NBS had higher mortality even when PIDD was ruled-out. Transient abnormal TREC-NBS was not associated with higher mortality, but unresolved or late abnormal TREC-NBS was associated with higher mortality. Infants with late abnormal TREC-NBS had severe prematurity, lower birth weight, lower Apgar scores, and higher percentage of congenital anomalies. Infants with abnormal TREC-NBS may be at a higher risk of morbidity and mortality and should be carefully followed, especially if discharged home before a repeat screen can be completed. This study explores the risk factors and mortality for newborns with secondary T-cell lymphopenia captured on T-Cell Receptor Excision Circles based newborn screening (TREC-NBS). Abnormal TREC-NBS allows for prompt life-saving interventions for primary immunological conditions such as Severe Combined Immunodeficiency (SCID), but can also be associated with non-immunologic conditions. Unresolved and late abnormal TREC-NBS is associated with higher mortality even without primary immunodeficiency, likely detected in infants with more severe prematurity, lower birth weight, and congenital anomalies. TREC-NBS positive infants with secondary T-cell lymphopenia require special attention and close monitoring.

FOXI3 haploinsufficiency contributes to low T-cell receptor excision circles and T-cell lymphopenia

Newborn screening can identify neonatal T-cell lymphopenia through detection of a low number of copies of T-cell receptor excision circles in dried blood spots collected at birth. After a positive screening result, further diagnostic testing is required to determine whether the subject has severe combined immunodeficiency or other causes of T-cell lymphopenia. Even after thorough evaluation, approximately 15% of children with a positive result of newborn screening for T-cell receptor excision circles remain genetically undiagnosed. Identifying the underlying genetic etiology is necessary to guide subsequent clinical management and family planning. We sought to elucidate the genetic basis of patients with T-cell lymphopenia without an apparent genetic diagnosis. We used clinical genomic testing as well as functional and immunologic assays to identify and elucidate the genetic and mechanistic basis of T-cell lymphopenia. We report 2 unrelated individuals with nonsevere T-cell lymphopenia and abnormal T-cell receptor excision circles who harbor heterozygous loss-of-function variants in forkhead box I3 transcription factor (FOXI3). Our findings support the notion that haploinsufficiency of FOXI3 results in T-cell lymphopenia with variable expressivity and that FOXI3 may be a key modulator of thymus development.

TREC/KREC Newborn Screening followed by Next-Generation Sequencing for Severe Combined Immunodeficiency in Japan.

The aim of this study is to evaluate the usefulness of T cell receptor excision circle (TREC) and/or kappa-deleting recombination excision circle (KREC) measurements integrated with diagnostic next-generation sequencing (NGS) analysis using a severe combined immunodeficiency (SCID) newborn screening (NBS) program. TREC and/or KREC values were measured in 137,484 newborns between April 2017 and December 2021 using EnLite TREC (n = 80,791) or TREC/KREC kits (n = 56,693). For newborns with positive screening results, diagnostic NGS analysis was performed with a 349-gene panel to detect genetic mutations associated with primary immunodeficiencies (PIDs). A total of 145 newborns (0.11%) had abnormal TREC and/or KREC values, and a genetic diagnosis was established in 2 patients with SCID (1 in 68,742 newborns) (IL2RG-SCID and reticular dysgenesis) and 10 with non-SCID PIDs with T and/or B cell deficiencies (1 in 13,748 newborns) using NGS analysis. Furthermore, TREC values of 2849 newborns were measured and confirmed the significant correlation between the results of both TREC and TREC/KREC kits (P < 0.001) and naïve T cell counts. We performed the first large-scale TREC and TREC/KREC NBS programs in Japan. Our NBS programs followed by the diagnostic NGS analysis for newborns with abnormal TREC and/or KREC values are useful for the early identification and rapid molecular evaluation of not only SCID but also different non-SCID PIDs.

Premature Infants Have Normal Maturation of the T Cell Receptor Repertoire at Term.

Premature infants are known to have immature immune systems compared to term infants; however, the impacts of ex utero immune development are not well characterized. Our previous retrospective clinical review showed prolonged T cell lymphopenia in a subset of extremely premature infants, suggesting that they may have lasting abnormalities in their T cell compartments. We used T cell receptor (TCR) repertoire sequencing to analyze the composition of the T cell compartment in premature and term infants in our NICU. We collected twenty-eight samples from individual subjects and analyzed the number of clonotypes, repertoire diversity, CDR3 length, and V gene usage between groups based on gestational age at birth and postmenstrual age at the time of sample collection. Further, we examined the TCR repertoire in infants with severe bronchopulmonary dysplasia (BPD) and those with abnormal T cell receptor excision circle (TREC) assays. Former extremely premature infants who were corrected to term postmenstrual age had TCR repertoire diversity that was more similar to term born infants than extremely premature infants, supporting normal maturation of the repertoire. Infants with severe BPD did not appear to have increased abnormalities in repertoire diversity. Decreased TCR repertoire diversity was associated with repeatedly abnormal TREC screening, although the diversity was within the normal range for subjects without low TRECs. This study suggests that extremely premature infants demonstrate normal maturation of the T cell repertoire ex utero. Further work is needed to better characterize postnatal T cell development and function in this population.

Implementation of Universal Newborn Screening for Severe Combined Immunodeficiency in Singapore While Continuing Routine Bacille-Calmette-Guerin Vaccination Given at Birth.

IntroductionSevere Combined Immunodeficiency (SCID) is generally fatal if untreated; it predisposes to severe infections, including disseminated Bacille-Calmette-Guerin (BCG) disease from BCG vaccination at birth. However, delaying BCG vaccination can be detrimental to the population in tuberculosis-endemic regions. Early diagnosis of SCID through newborn screening followed by pre-emptive treatment with anti-mycobacterial therapy may be an alternative strategy to delaying routine BCG vaccination. We report the results of the first year of newborn SCID screening in Singapore while continuing routine BCG vaccination at birth.MethodNewborn screening using a T-cell receptor excision circle (TREC) assay was performed in dried blood spots received between 10 October 2019 to 9 October 2020 using the Enlite Neonatal TREC kit. Patients with low TREC had lymphocyte subset analysis and full blood count performed to determine the severity of lymphopenia and likelihood of SCID to guide further management.ResultsOf the 35888 newborns screened in 1 year, no SCID cases were detected, while 13 cases of non-SCID T-cell lymphopenia (TCL) were picked up. Using a threshold for normal TREC to be >18 copies/μL, the retest rate was 0.1% and referral rate to immunologist was 0.04%. Initial low TREC correlated with low absolute lymphocyte counts (ALC), and subsequent normal ALC corresponded with increases in TREC, thus patients with normal first CD3+ T cell counts were considered to have transient idiopathic TCL instead of false positive results. 7/13 (54%) had secondary TCL (from sepsis, Trisomy 21 with hydrops and stoma losses or chylothorax, extreme prematurity, or partial DiGeorge Syndrome) and 6/13 (46%) had idiopathic TCL. No cases of SCID were diagnosed clinically in Singapore during this period and for 10 months after, indicating that no cases were missed by the screening program. 8/9 (89%) of term infants with abnormal TREC results received BCG vaccination within the first 6 days of life when TREC and ALC were low. No patients developed BCG complications after a median follow-up of 17 months.ConclusionNewborn screening for SCID can be implemented while continuing routine BCG vaccination at birth. Patients with transient TCL and no underlying primary immunodeficiency are able to tolerate BCG vaccination.

Ten Years of Newborn Screening for Severe Combined Immunodeficiency (SCID) in Massachusetts

To examine the outcomes of population newborn screening (NBS) for severe combined immunodeficiency (SCID) by using T-cell receptor excision circles (TRECs) after the first decade of implementation in Massachusetts.Infants born in Massachusetts screened through the New England Newborn Screening Program between February 1, 2009, and January 31, 2019.The authors present a population cohort of infants receiving NBS for SCID, both before and after mandatory NBS implementation in 2016.Of 720 038 infants screened, normal TRECs were identified in 99.71%. At least 1 abnormal specimen was identified in 2072 infants (0.29% of those screened). Of these infants, 1771 had an additional TREC result, which was normal, and were released from the screening program (83.8% of those with an abnormal TREC result before confirmatory flow cytometry and 1.7% after further lymphocyte phenotyping). Further evaluation by flow cytometry occurred in 237 infants (11.4% of those with at least 1 abnormal TREC result), and low naïve T-cell percentages were associated with a greater risk of a combined immunodeficiency. A total of 9 infants were diagnosed with SCID or leaky SCID, 7 were diagnosed with other combined immunodeficiencies, and 3 were diagnosed with athymia (0.4%, 0.3%, and 0.1%, respectively. of those with an abnormal TREC screen). The incidence of SCID or leaky SCID was reported as ∼1 in 80 000; however, more broadly, the incidence of severe T-cell lymphopenia for which definitive treatment was indicated was ∼1 in 50 000 infants. All patients with SCID or leaky SCID underwent hematopoietic cell transplant or gene therapy for which survival was 100%, and 1 patient with athymia underwent a successful thymus transplant. With NBS, the researchers successfully identified all known cases of SCID. Compared with survival before NBS by TREC, survival improved significantly for infants with SCID or leaky SCID (100% [n = 9] vs 57% [n = 7]. Of 162 infants with T-cell counts <2500 but above 1500 µL, no cases of SCID were identified, but 8 infants were diagnosed with partial DiGeorge syndrome.NBS of TREC levels is highly sensitive and specific, with the ability to improve early diagnosis of SCID. When compared with historical rates of SCID diagnosis and management before TREC evaluation by NBS, screening can reduce the time to transplant (3.4 months versus 6.5 months) and improve outcomes for infants with SCID.Screening for SCID has been a significant addition to NBS programs across the country, and multiple studies have suggested this to be a cost-effective strategy. As of December 2018, all 50 states have begun to screen for SCID at birth. SCID is often unrecognized in early life, but survival improves with early diagnosis. With this study, the authors provide long-term outcomes and highlight the benefit of these programs, suggesting a 43% reduction in mortality from a condition that can be successfully treated when identified early. From these data, the sensitivity, specificity, negative predictive value, and positive predictive value can be calculated (100%, 99.7%, 100%, and 0.91%, respectively). Notably, despite high sensitivity and specificity, a population prevalence of ∼1:50 000 to 80 000 lowers the positive predictive value of screening. Although early referral to an immunologist is important in infants with abnormal TREC results, these data also suggest the absolute risk of SCID in these infants is low (<1%). Thus, when counseling families of infants with an abnormal TREC screen, there is room for cautious optimism, pending results of definitive testing. More work is needed to better understand the short- and longer-term psychosocial impacts of a false-positive NBS result for SCID on families with unaffected infants after a positive screening test result.

Ten Years of Newborn Screening for Severe Combined Immunodeficiency (SCID) in Massachusetts

Massachusetts began newborn screening (NBS) for severe combined immunodeficiency (SCID) using measurement of T-cell receptor excision circles (TRECs) from dried blood spots. We describe developments and outcomes from the first 10 years of this program (February 1, 2009, to January 31, 2019). TREC values, diagnostic, and outcome data from all patients screened for SCID were evaluated. NBS of 720,038 infants prompted immunologic evaluation of 237 (0.03%). Of 237, 9 were diagnosed with SCID/leaky SCID (4% of referrals vs 0.001% general population). Another 7 were diagnosed with other combined immunodeficiencies, and 3 with athymia. SCID/leaky SCID incidence was approximately 1 in 80,000, whereas approximately 1 in 51,000 had severe T-cell lymphopenia for which definitive treatment was indicated. All patients with SCID/leaky SCID underwent hematopoietic cell transplant or gene therapy with 100% survival. One patient with athymia underwent successful thymus transplant. No known cases of SCID were missed. Compared with outcomes from the 10 years before SCID NBS, survival trended higher (9 of 9 vs 4 of 7), likely due to a lower rate of infection before treatment. Our data support a single NBS testing-and-referral algorithm for all gestational ages. Despite lower median TREC values in premature infants, the majority for all ages are well above the TREC cutoff and the algorithm, which selects urgent (undetectable TREC) and repeatedly abnormal TREC values, minimizes referral. We also found that low naïve T-cell percentage is associated with a higher risk of SCID/CID, demonstrating the utility of memory/naïve T-cell phenotyping as part of follow-up flow cytometry.

A Novel De Novo PGM3 Pathogenic Mutation Identified in an Infant Presenting with Abnormal TREC Assay and Severe Neutropenia

Phosphoglucomutase 3 (PGM3) deficiency is an autosomal recessive congenital glycosylation disorder associated with a variable clinical phenotype including immunodeficiency, atopy and progressive bone marrow failure. We present an infant with compound heterozygous pathogenic PGM3 mutations, including a novel de novo variant, who initially presented with an abnormal T-cell receptor excision circles (TREC) assay on newborn screen.

When Screening for Severe Combined Immunodeficiency (SCID) with T Cell Receptor Excision Circles Is Not SCID: a Case-Based Review.

Newborn screening efforts focusing on the quantification of T cell receptor excision circles (TRECs), as a biomarker for abnormal thymic production of T cells, have allowed for the identification and definitive treatment of severe combined immunodeficiency (SCID) in asymptomatic neonates. With the adoption of TREC quantification in Guthrie cards across the USA and abroad, typical, and atypical SCID constitutes only ~ 10% of cases identified with abnormal TRECs associated with T cell lymphopenia. Several other non-SCID-related conditions may be identified by newborn screening in a term infant. Thus, it is important for physicians to recognize that other factors, such as prematurity, are often associated with low TRECs initially, but often improve with age. This paper focuses on a challenge that immunologists face: the diagnostic evaluation and management of cases in which abnormal TRECs are associated with variants of T cell lymphopenia in the absence of a genetically defined form of typical or atypical SCID. Various syndromes associated with T cell impairment, secondary forms of T cell lymphopenia, and idiopathic T cell lymphopenia are identified using this screening approach. Yet there is no consensus or guidelines to assist in the evaluation and management of these newborns, despite representing 90% of the patients identified, resulting in significant work for the clinical teams until a diagnosis is made. Using a case-based approach, we review pearls relevant to the evaluation of these newborns, as well as the management dilemmas for the families and team related to the resolution of genetic ambiguities.

Functional Confirmation of DNA Repair Defect in Ataxia Telangiectasia (AT) Infants Identified by Newborn Screening for Severe Combined Immunodeficiency (NBS SCID)

The introduction of newborn screening for severe combined immunodeficiencies (NBS SCID) in 2010 was a significant public health milestone. Although SCID was the primary target, several other conditions associated with severe T-cell lymphopenia have subsequently been identified as secondary targets. The differential diagnosis in infants with an abnormal T-cell receptor excision circle result on NBS SCID who do not meet criteria for typical SCID is often broad, and often the evaluation of these conditions requires immunological and functional testing, in conjunction with genetic analysis, to obtain an accurate diagnosis and develop an appropriate management and treatment plan. We describe here 3 infants identified by NBS SCID, who required additional workup as they did not have a typical SCID phenotype and meet the relevant diagnostic criteria. Genetic testing identified pathogenic variants in ATM in all 3 patients, and the pathogenicity of the variants was confirmed by a functional flow cytometry assay. The patients underwent immunological and genetic workup to identify an underlying cause of their abnormal NBS SCID. Ataxia telangiectasia (AT) was suspected based on clinical and family history, and immunological analyses. The diagnosis was confirmed in all patients with a rapid functional flow cytometric assay and genetic testing. A rapid functional flow cytometry assay was used as a diagnostic and confirmatory tool, in conjunction with genetic testing, to make a diagnosis of AT. Experimental validation of the causal relationship between genotype and phenotype allowed for expeditious diagnosis, which facilitated early discussions with families regarding prognosis, treatment, and management. Even with increased rapidity and access to genetic results, functional testing is required for clinical diagnosis in infants identified by NBS SCID who do not fit into the classic categories or have novel genetic variants to confirm the diagnosis. Consideration should be given to the use of functional assays as an essential component of an integrated evaluation to characterize the genetics and mechanisms of inborn errors of immunity.

Retrospective Analysis of a New York Newborn Screen Severe Combined Immunodeficiency Referral Center.

In 2010, the New York State (NYS) Newborn Screen (NBS) Program added the T cell receptor excision circle (TREC) assay to screen for severe combined immunodeficiency disorder (SCID). The objective of this study was to perform a retrospective chart review of 199 infants referred to a single institution for abnormal TREC on NYS NBS between 2010 and 2017. Statistical analysis included analysis of variance, logistic regression models, chi-square, and linear mixed models. One hundred ninety-nine infants were found to have a TREC value of fewer than 200 copies/μL on NYS NBS. Infants were stratified as primary immunodeficiency (PID) (n= 54), immunocompetent (n= 133), lost to follow-up (n= 8), or deceased (n= 4). PID included SCID (n= 3), DiGeorge (n= 6), idiopathic lymphopenia (IL) (n= 44), and other syndromes associated with lymphopenia (n= 3). The 3 SCID cases were identified and brought to treatment, although all experienced significant infections. The study population was found to be predominately non-Hispanic, African American, and male. There was a difference in the average TREC values among those with immunocompetence (83 copies/μL), IL (81 copies/μL), and PID (40 copies/μL) (p< 0.05). On follow-up of 40 patients with IL, patients typically did not have severe infections during first few years of life. This study demonstrates that TREC value can be used to stratify infants for further confirmatory testing to exclude PID. Risk factors, such as stressful prenatal/postnatal conditions, prematurity, race, and sex may affect TREC value but cannot explain all causes of lymphopenia. This study may assist providers in risk stratifying the likelihood of PID with an abnormal TREC and determining the extent of the initial work up that is necessary at the time of a newborn's presentation.

Abnormal Newborn Screening Follow-up for Severe Combined Immunodeficiency in an Amish Cohort with Cartilage-Hair Hypoplasia

Cartilage-hair hypoplasia (CHH) is an autosomal recessive, short limb skeletal dysplasia with a variable immunologic phenotype. The spectrum of immune function ranges from clinically normal to severe combined immunodeficiency (SCID). Multiple studies have shown that abnormal immune parameters may not predict severe outcomes. Newborn screening (NBS) using T cell receptor excision circle (TREC) assay can now effectively identify infants with severe T cell deficiency who are at risk for SCID. NBS has allowed for cost-effective identification of patients with SCID and improved outcomes with hematopoietic stem cell transplant (HSCT). Ohio reports two abnormal TREC results: decreased and absent TREC. This study evaluated the laboratory and clinical differences in eight Amish patients with CHH with an abnormal TREC result on the NBS. There were four patients with absent TREC and four patients with decreased TREC. The absent TREC patients had lower CD3, CD4, naïve CD4, CD8 cells, and phytohemagglutinin (PHA)-induced lymphocyte proliferation. Three patients with absent TREC were diagnosed with SCID and two underwent successful HSCT. Patients with absent TREC experienced more CHH-related morbidity including anemia requiring transfusion, Hirschsprung's disease, and failure to thrive. No patients with decreased TREC required HSCT. Our study indicates that CHH patients with absent TREC tend to have more severe immunological and clinical phenotype than patients with decreased TREC. Confirmation of these trends in a larger group would guide providers and parents in a timely referral for HSCT, or cost-effective surveillance monitoring of children with a life-threatening illness.

Newborn Screening for Severe Combined Immunodeficiency and T-cell Lymphopenia in California, 2010-2017.

Newborn screening for severe combined immunodeficiency (SCID) was instituted in California in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from dried blood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC results were followed-up with liquid blood testing for T-cell abnormalities. We report the performance of the SCID screening program and the outcomes of infants who were identified. Data that were reviewed and analyzed included demographics, nursery summaries, TREC and lymphocyte flow-cytometry values, and available follow-up, including clinical and genetic diagnoses, treatments, and outcomes. Infants with clinically significant T-cell lymphopenia (TCL) were successfully identified at a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95% confidence interval 1 in 51 000-1 in 90 000) were found. Prompt treatment led to 94% survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including 4 with complete DiGeorge syndrome who received thymus transplants. Although no cases of typical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID had normal neonatal TREC screens but came to clinical attention at 7 and 23 months of age. Population-based TREC testing, although unable to detect immune defects in which T cells are present at birth, is effective for identifying SCID and clinically important TCL with high sensitivity and specificity. The experience in California supports the rapid, widespread adoption of SCID newborn screening.

ABNORMAL NEWBORN SCREEN IN A WHIM SYNDROME PATIENT

Introduction Newborn screen (NBS) for primary immunodeficiency is performed in the vast majority of states. Dried blood spots are collected from infants. The samples undergo quantitative PCR to quantify T cell receptor excision circles (TRECs). This screening detects Severe Combined Immunodeficiency (SCID) as well as hypomorphic mutations in SCID genes, conditions resulting in the arrest of T cell development such as DiGeorge syndrome and conditions presenting with T cell lymphopenia. Case Description A full term infant was born to a mother with known WHIM syndrome. He was admitted to the NICU on his first day of life for fever, tachypnea and hypoxia. Blood and cerebrospinal fluid cultures were negative at 48 hours and the patient's symptoms resolved. Echocardiogram demonstrated patent ductus arteriosus, pulmonary artery stenosis and atrial septal defect versus patent foramen ovale. On NBS TRECs were not detected until 34 cycles of PCR amplification. ALC was 600 cells/microliter. Flow cytometry showed T and B cell lymphopenia. ANC was initially 9,000 cells/microliter in the setting of fever. At 5 months of life ANC was only 32 cells/microliter. Genetic testing performed at the National Institutes of Health confirmed a heterozygous CXCR4 mutation, specifically c.1000C>T (p.Arg334Ter) consistent with WHIM syndrome. Prophylactic antibiotics, replacement immunoglobulin, granulocyte colony-stimulating factor and CXCR4 antagonists are considerations for therapy. Discussion Low TRECs on NBS may be indicative of disease with life-threatening complications. Immunologists should consider WHIM syndrome when evaluating a patient with abnormal TRECs on NBS. Although this condition is rare, this case expands the differential diagnosis of an abnormal NBS.

ABNORMAL TRECS AND SEVERE LYMPHOPENIA SECONDARY TO MATERNAL USE OF AZATHIOPRINE

Introduction T-cell receptor excision circles (TREC) analysis is an important addition to the newborn screen (NBS) for early identification of primary immune deficiency disorders (PID), specifically severe combined immune deficiency (SCID). TREC screening has also identified T-cell lymphopenias secondary to causes not associated with PID. Our case illustrates a neonate whose profound lymphopenia was likely caused by in-utero exposure to an immunomodulatory medication. Case Description A term male infant, born to a mother on azathioprine for systemic erythematous lupus, was referred for abnormal TRECs on NBS. Flow cytometry revealed marked lymphopenia affecting T, B and NK cell lines (338/mm3, 1/mm3 and 9/mm3, respectively). Flow cytometry showed 56% CD3/CD4/RA+ cells, higher than expected in SCID, yet below normal for age. Repeat flow cytometry one week later demonstrated slightly improved CD3/4/8 counts, ongoing severe B cell lymphopenia, with normalization of NK cell counts. Genetic testing for 207 known PID mutations was non-diagnostic. Three weeks later, B-cell counts normalized, and T-cell counts dramatically improved. Placental transfer of azathioprine was felt to be responsible for the previous lab abnormalities. Discussion In addition to SCID, transient T-cell lymphopenias of unclear clinical significance are identified by TREC screening. It remains uncertain which of these patients warrant further work up, and infants and their families may be subject to unnecessary testing and emotional stress. This case is a reminder that the family medical history is crucial in identifying possible secondary causes of abnormal TRECs. It also raises demonstrates the potential impact of maternal immunosuppression on infant hematopoiesis.

NEW PATHOGENIC VARIANT ON IL-7R GENE ASSOCIATED WITH SCID

Introduction T-B+NK+ Severe Combined Immunodeficiency (SCID) due to mutations in the genes encoding the IL-7 receptor represent about 10% of American SCID cases. We report a patient found to have a c.675C>A (p.Tyr225*) homozygous mutation in exon 5. This variant is not present in population databases, and has not been reported in the literature in individuals with IL-7 receptor disease. Case Description Our medical center received a referral for abnormal T-cell receptor excision circle (TREC) results on a newborn screen. The patient's mother had an uncomplicated pregnancy, and there was no consanguinity or family history concerning for a primary immune deficiency. Lymphocyte subsets revealed an absence of T cells, along with present, but diminished B-cells and Natural-Killer cells. The patient had an absent thymic shadow on chest X-ray. He was also found to have poor mitogenic responses. FISH for DiGeorge syndrome was negative for deletions in 22q11.2 and 22q13, and testing for ADA deficiency was negative. Genetic testing resulted in a c.675C>A (p.Tyr225*) homozygous mutation. The patient has had a confirmed infection with Rhinovirus. He is currently on bacterial and fungal prophylaxis, and receiving immune globulin while awaiting bone marrow transplantation. Discussion A homozygous mutation in IL7R, Exon 5, c.675C>A (p.Tyr225*) creates a premature translational stop signal in the IL7R gene, resulting in an absent or disrupted protein product. Loss of function variants of IL7R are known to be pathogenic. This case reinforces the need for researchers to functionally evaluate all mutations found in patients.

ABNORMAL TREC RESULTS ON THE OHIO NEWBORN SCREEN IN CHILDREN WITH CARTILAGE-HAIR HYPOPLASIA

Introduction While newborn screening (NBS) using T-cell receptor excision circle (TREC) analysis was introduced primarily to screen for severe combined immunodeficiency (SCID), it also identifies T-cell lymphopenia or low naive T-cell numbers due to other etiologies. Cartilage-hair hypoplasia (CHH) is an autosomal recessive disorder caused by mutations in the RMRP gene that result in short limbed skeletal dysplasia and a range of immunologic deficits involving both cellular and humoral immune function. The immune defect can range from minor abnormalities to a SCID phenotype. Methods We analyzed the NBS TREC results performed in Ohio between July 2013 and July 2018. We reviewed the results of the immunologic workup in patients with abnormal TREC results who were later confirmed to have CHH. Results A total of 693,706 unique infants had NBS performed between July 2013 and July 2018, with 192 (0.027%) having abnormal TREC results. Eight infants with abnormal TREC results were diagnosed with CHH. This included 6 cases of non-SCID T-cell lymphopenia and 2 children with SCID phenotypes. In the children with abnormal screening and a diagnosis of CHH, the absolute CD4 lymphocyte counts on initial immunophenotyping ranged from 76 – 757 cells/mm3 (median = 552 cells/mm3). The two CHH patients with SCID received bone marrow transplants. Conclusions CHH is a rare genetic disorder associated with widely varying levels of T-cell lymphopenia ranging from clinically insignificant to SCID. The T-cell lymphopenia may cause an abnormal result on the NBS TREC analysis, allowing for early diagnosis of a significant immunodeficiency and institution of life-saving therapies.

Newborn Screening for Severe Combined Immunodeficiency.

This review provides a brief history of newborn screening (NBS) for severe combined immunodeficiency (SCID), discusses the theoretical basis for the T cell receptor excision circle (TREC) assay, highlights the results of recent studies using the TREC, and provides practical advice for the evaluation of infants with an abnormal TREC assay. Currently, all but three states perform NBS for SCID in the USA. NBS using the TREC assay is highly sensitive in identifying infants with SCID and may also identify infants with T cell lymphopenia due to other causes such as congenital syndromes, multiple congenital anamolies, and some combined immunodeficiencies. Regardless of the genetic etiology, all forms of SCID are characterized by a severe deficiency of naïve T cells. TRECs are a biomarker of newly formed, naïve T cells that have recently left the thymus. Consequently, the TREC assay identifies infants with SCID and other causes of severe T cell lymphopenia.

Diagnostic Challenges in the Era of Genomic Sequencing and Newborn Screening

Early diagnosis of rare immune disorders is important for clinical care and of great interest for the study of immune pathways. Severe combined immunodeficiency (SCID) is a collective term for the most profound inherited defects of T cell development combined with B cell defects or dysfunction. While fatal without treatment, SCID is treatable by allogeneic hematopoietic cell transplantation, or in certain genotypes by enzyme or gene therapy. Avoidance of life-threatening infections to provide optimal treatment and outcomes for affected infants has led to population-based SCID newborn screening (NBS). Infants with SCID fail to generate a diverse repertoire of functional T cells, and consequently have very low numbers of T cells and T cell receptor excision circles (TRECs), DNA byproducts of T cell receptor gene rearrangement. TRECs are readily measured in DNA isolated from newborn dried blood spots (DBS) collected for population based screening. Thus newborn screening for insufficient TRECs identifies SCID before infections occur. As SCID NBS has become widespread, new disease definitions are required for healthy-appearing affected infants without failure to thrive or opportunistic infections. Typical SCID cases have <300 autologous T cells/uL, <10% of the lower range of normal proliferation to the mitogen phytohemmaglutinin A, and/or detectable maternal T cell engraftment, most often with deleterious mutations in recognized SCID genes. One fourth of all SCID cases are “leaky” due to hypomorphic SCID gene mutations; these cases are also detected by TREC testing; they may have >300 T cells/uL, but have impaired T cell function and lack naïve CD4 T cells expressing CD45RA. A subset of infants with leaky SCID have Omenn syndrome, with expansion of oligoclonal, dysregulated T cells leading to adenopathy, erythroderma, hepatosplenomegaly, eosinophilia, and elevated IgE. In addition to these primary target disorders of population screening for SCID, the TREC test identifies infants with additional conditions due to either impaired production or increased loss of T cells. Non-SCID congenital syndromes with variable degrees of T cell lymphopenia (TCL) include DiGeorge syndrome/22q11.2 deletion, CHARGE syndrome, trisomy 21, and ataxia telangiectasia, among others. TREC NBS also finds infants with secondary TCL, in which T cell generating capacity is intrinsically normal, but circulating T cells are diminished as a consequence of other factors, including hydrops, congenital heart disease, chylothorax, neonatal leukemia, maternal immunosuppressive medications taken during pregnancy, or extreme preterm birth. The T cells of these infants normalize once their primary problems resolve. A particularly challenging group of infants are those with abnormal TREC screen results and non-SCID TCL, but no immediate diagnosis. About half of these have syndromes, such as DiGeorge/22q deletion, but with mild or initially unapparent features; others experience resolution of TCL over time, while still others prove to have previously unknown immune disorders that may be diagnosed after deep sequencing and research functional studies. It is important to remember that many serious disorders of T cells are not identified by TREC screening if the block in T cell development or function occurs at a later stage than T cell receptor rearrangement; combined immunodeficiencies (CIDs) with TRECs that are often normal include Zap-70 deficiency and MHC class I and II non-expression. Thus, while virtually completely sensitive and highly specific for the intended target, SCID, population based TREC screening leaves us with both diagnostic dilemmas presented by non-symptomatic infants with low T cells and inability to capture individuals with the >300 non-SCID primary immunodeficiency disorders that would also benefit from early intervention. Deep sequencing is not yet clinically useful, not only due to cost, turnaround time and technical limitations, but primarily due to problems of interpretation, given the extraordinary number of genomic variants of uncertain significance. DisclosuresPuck:InVitae, a clinical DNA sequencing company: Other: Spouse employment and stock options; UpToDate: Patents & Royalties: Recieve royalties to write and edit entries on primary immune disorders.

P189 ATM protein, DOCK8, and ZAP70 heterozygous mutations presenting as abnormal T-cell receptor excision circle values

Abnormal T-cell receptor excision circle (TREC) values on a newborn screen (NBS) can reflect severe combined immunodeficiency (SCID) or another form of T cell lymphopenia. We report a case of multiple heterozygous mutations in the absence of T cell lymphopenia that resulted in low TREC values on NBS.