Neonatal Screening for Severe Combined Immunodeficiency (SCID) by TREC &KREC assay
Content Outline
T&B cell REARANGMENT
T-cell Receptor Excision Circles (TREC)
δ Rec single joint TREC
kappa-deleting recombination excision circles (KRECs)
Types of immunodeficiency diseases
Primary immunodeficiency diseases)B &T cells)
Severe Combined Immunodeficiency (SCID)
SCID disorders effects on immunologic mechanism
Essential tool for diagnosis of primary immune deficiencies (PIDs)
TREC test for SCID
Content outline
Evaluation of TREC
Explain why quantifying T-cell receptor excision circles (TREC) is utilized to detect newborns with SCID
Describe quantitative real-time PCR assay for TREC
TREC concentrations in healthy and NICU babies
TREC concentrations vs birth weight
Wisconsin algorithm
Answer to this question that How can SCID be prevented?
Treatment of SCID disease
Discussion
overview
Reference
T&B CELL RECEPTOR
T CELL REARANGMENT
B CELL REARANGMENT
TREC
The excised DNA fragments of the TCR that are not destined to be incorporated into a recombinant receptor gene are also ligated at their ends, forming a variety of circular DNA by-products called T-cell receptor excision circles (TRECs).
δ Rec single joint TREC
Circular DNA formed as a byproduct of successful T-cell receptor rearrangement (TCR alpha chain rearrangement, deleting the delta locus)
One particular circular species—the δrec-ψJα TREC—is produced late in maturation by 70% of all T cells that are destined to express αβ TCRs.
TRECs are stable but are not replicated during mitosis . Therefore, they persist but become diluted as T cells are dividing.
Thus, the number of TREC copies can be used as an indicator of thymic production of naïve T cells, with a low TREC number signaling concern for inadequate autologous T-cell production.
In the bone marrow, progenitor B cells undergo V(D)J rearrangements of the Ig heavy chain (IGH) locus followed by those of the light chains (IGK and IGL),After successful IGH rearrangements at the Pre-B stage, a VJ recombination on the IGK locus is initiated. If it is not productive, another recombination between the Ig kappa deleting element or like (IGKDEL) and one of the upstream recombination signal sequences (RSS) renders the IGK allele non-functional.
In 30%-50% of cases, this occurs through the intron RSS-IGKDEL rearrangement, by which the IGKC exon and its enhancers (iE κ, 3’Eκ) are excised, with the creation of the so-called kappa-deleting recombination excision circles (KRECs).
kappa-deleting recombination excision circles (KRECs)
KRECs carry an intron RSS-IGKDEL SJ, and remain in the cells, but, as they cannot be replicated, they will be diluted during peripheral expansion of mature B cells. Instead, an intron RSS-IGKDEL CJ is formed and stably retained in the genomic DNA , because, due to the enhancer loss, any further rearrangement in the IGK locus is precluded.
KREC level has been evaluated only very recently in children with PID , mainly in patients affected by X linked or non X-linked agammaglobulinemia (XLA and non-XLA, respectively), and it used for those with CVID.
Types of immunodeficiency diseases
Primary
Heterogeneous group of rare, single-gene diseases leading to impairment of the immune responses
Inherited as X-linked or autosomal disorders, either dominant or recessive
gene defects lead to missing enzymes, developmental arrest in immune differentiation, absent or non-functional proteins, abnormal DNA repair, altered signal transduction, impairment of cell-to-cell and intracellular communications
Secondary
Systemic disorders (e.g., diabetes, undernutrition, HIV infection)
Immunosuppressive treatments (e.g., cytotoxic chemotherapy, bone marrow ablation before transplantation, radiation therapy)
Prolonged serious illness
SCID disease???? bubble boy disease
David Vetter, the famous “bubble boy” of the 1970s, had to live in a plastic pod , eating sterilized food and wearing sterilized clothes, to avoid the everyday germs that can be fatal to those with IL2RG deficiency He lived in a sterile plastic pod from birth until he died at age 12.
Heterogeneous group of genetic defects with an overall incidence of about 1 in 40,000 to 75,000 newborns.
Characterized by poor T cell production and failure of B cells to generate protective antibodies.
Most frequently observed in the first few months of life and the median age at diagnosis is 4-7 months.
Treated by Hematopoietic cell transplantation (HCT), adenosine deaminase (ADA) enzyme replacement, or gene therapy.
Early detection of SCID is a key to successful treatment.
SCID and CID disorders based on immunologic mechanism and expectation to have abnormally low TRECs at birth
essential tool for diagnosis of primary immune deficiencies (PIDs)
Flow cytometry is an essential tool in the diagnosis of primary immune deficiencies (PIDs). Lymphocyte subset enumeration can be determined by measuring expression of certain cell surface molecules.
Flow cytometry using markers specific for T cells (CD3), B cells (CD19), or NK cells (CD56) is the first step in evaluating children with suspected SCID due to clinical manifestations or a failed newborn screen for SCID.
There are several PIDs where it is possible to assess specific protein expression, either as a screening test for phenotype correlation or as a confirmatory test. Four specific examples are provided to discuss the use of specific protein analysis in diagnosis of PIDs: Btk protein expression in X-linked agammaglobulinemia (XLA), CD40 ligand (CD40L) expression in X-linked hyper-IgM syndrome, LRBA (lipopolysaccharide-responsive beige-like anchor) protein expression in LRBA deficiency, and DOCK8 (dedicator of cytokinesis 8) protein expression in DOCK8 deficiency.
TREC test for SCID
On January 1, 2008, Wisconsin (USA) became the first state in the world to screen all newborns for SCID through a method based on measurement of T cell receptor excision circles (TRECs) by polymerase chain reaction (PCR), using DNA extracted from newborn dried blood spots (Guthrie cards).
TRECs were initially used to monitor generation of new T cells in HIV-infected individuals receiving effective anti retroviral treatment.
protocol
The TREC assay was adapted to NBS as a real time polymerase chain reaction(PCR)that amplifies DNA extracted from DBS to detect TRECs and KREC as a biomarker of T and B cells.
A quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood.
Absent or few TRECs are found in infants who have an inadequate number of naïve T cells from any cause.
Real-time Quantitative Polymerase Chain Reaction (TaqMan)
Amplify and quantify TREC DNA
Amplify and quantify reference gene DNA
Plasmids are used as standards
TaqMan Probe
– sequence specific
– fluorescent signal
TREC Concentrations
How can SCID be prevented?
. In general, SCID is a genetic condition that is not the result of an environmental toxin or induced by a pathogen.
. Single gene mutations are sporadic and occur in essentially every ethnic group. Consanguinity increases the risk for SCID, but many forms are also the result of spontaneous mutations.
treatment
Isolation
Your baby may need to stay away from young children. This makes it less likely he or she will catch an illness from another child.
Immunoglobulin Replacement Therapy
Babies with SCID should be placed on immunoglobulin replacement therapy if they are more than three months of age or have already had infections. Regular immunoglobulin replacement therapy can replace missing antibodies that help your baby fight infections.
Bone Marrow Transplant
The most effective treatment for SCID is a bone marrow transplant. Bone marrow makes cells for fighting infections and illnesses. In a bone marrow transplant, bone marrow cells from a person with a working immune system are given to a person with SCID, whose bone marrow cannot make cells to fight infections and illnesses.
discussion
In recent years, effective incorporation of TREC based NBS for SCID was reported in several studies. Techniques for measuring TRECs and specific screening and referral algorithms vary between states and countries that have reported successful implementation of SCID NBS.
Thus, the test detects any genetic causes of SCID or impaired T-cell production as well as conditions in which there is abnormal loss of T cells from the peripheral circulation. If this TREC assay do in early neonatal babies ,its possible Treatment some patients.
Recently an infant with SCID has been identified by newborn screening in Massachusetts and the U.S. Department of Health and Human Services recommended the addition of SCID to the uniform screening panel for all newborns.
overview
Definition
Circular DNA formed as a byproduct of successful T-cell receptor rearrangement, which occurs in the thymus
Characteristics
Present within CD4+ and CD8+ T-cells 1 or 2 copies per cell (initially) Do not replicate during mitosis
Screening Test
T- Cell Receptor Excision Circle (TREC)
Treatment
hematopoietic stem cell transplant (HSCT), enzyme replacement, gene therapy
•SCID is fatal unless treated
•Prompt treatment for SCID infants significantly increases survival
•SCID infants do not have any symptoms at birth, making prompt diagnosis difficult
•T-cell receptor excision circles (TREC) assays are currently being used to screen newborns for SCID
•TRECs are not specific for SCID, but markers for T-cell Lymphocytopenia, recent thymic emigrant T-cells
•Cut-off for TREC concentrations vary from state to state and are method dependent
•Screening for SCID with TREC – 100% sensitivity 98% specificity
Take Home Points
Reference
1. Chase, N.M., J.W. Verbsky , and J.M. Routes, Newborn screening for SCID: three years of experience. Ann N Y Acad Sci, 2011. 1238: p. 99-105.
2. Puck, J.M., The case for newborn screening for severe combined immunodeficiency and related disorders. Annals of the New York Academy of Sciences, 2011. 1246: p. 108-117.
3. Buelow, B.J., J.M. Routes, and J.W. Verbsky, Newborn screening for SCID: where are we now? Expert Rev Clin Immunol, 2014. 10(12): p. 1649-57.
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5. Adams, S.P., et al., Screening of neonatal UK dried blood spots using a duplex TREC screening assay. J Clin Immunol, 2014. 34(3): p. 323-30.
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7. Kwan, A., et al., Newborn Screening for Severe Combined Immunodeficiency in 11 Screening Programs in the United States. JAMA, 2014. 312(7): p. 729-738.
8. Lynch, H.E. and G.D. Sempowski, Molecular Measurement of T cell Receptor Excision Circles. Methods in molecular biology (Clifton, N.J.), 2013. 979: p. 147-159.
Kwan, A. and J.M. Puck, History and current status of newborn screening for severe combined immunodeficiency. Semin Perinatol, 2015. 39(3): p. 194-205.
Cossu, F., Genetics of SCID. Italian Journal of Pediatrics, 2010. 36: p. 76-76.
Serana, F., et al., Use of V(D)J recombination excision circles to identify T- and B-cell defects and to monitor the treatment in primary and acquired immunodeficiencies. J Transl Med, 2013. 11: p. 119.