In the germ line and all non-lymphoid cells, the gene loci encoding antibody molecules and T cell antigen receptor chains are composed of large numbers of alternate segments, called variable (V), diversity (D) and joining (J) regions that lie upstream of constant regions. Recombination of the T cell receptor (TCR) genes in the thymus is the process whereby a diverse repertoire of T cells is generated from randomly chosen alternate sections to synthesize a unique rearrangement in each cell. In the TCRα locus a Vα segment becomes linked to a Jα and a Cα segment through cutting and rejoining of the DNA by a series of enzymes that produce double strand DNA breaks at specific sites and then carry out processing and repair. Only T cell progenitors in which re-ligation produces an in-frame, rearranged locus are selected to survive and mature. The excised DNA fragments that are not destined to be incorporated into the mature TCR locus can be joined at their ends to form a great variety of circular DNA byproducts, called T cell receptor excision circles, or TRECs. Late in the maturation program, 70% of the thymocytes that will ultimately express αβ T cell receptors form one specific circular DNA TREC, the δRec-ψJa signal joint TREC, from the excised TCRδ gene (TCRD) that lies within the TCRα gene (TCRA) locus (). The DNA circles are stable and are maintained following cell divisions, but because they do not replicate they become diluted as T cells proliferate by mitotic division. A quantitative PCR reaction across the joint of the circular DNA, using primers indicated by the short arrows in , provides the TREC copy number, an indicator of new thymic emigrant T cells being produced.
Figure 1 Generation of the δRec-ΨJα TREC. The germ line configuration of the TCRA locus, with TCRD embedded, is shown at the top of the Figure, which also shows the points (gray dots) at which the DNA is cut to excise the TCRD locus in (more ...)
The groups of van Dongen et al.48
and Douek et al.49
found that TRECs are a biomarker for newly produced, naïve T cells that emigrate into peripheral blood from the thymus. Douek noted that infant blood samples have the highest numbers of TRECs, around one TREC per 10 T cells, reflecting the high rate of new T cell generation early in life. Older children and adults have 10-fold and 100-fold lower TRECs, respectively, reflecting increased peripheral T cell expansion as compared to new production of naïve T cells. TREC copy number varies widely between individuals, so a single measurement is not a useful clinical test in most settings, although serial measurements in a patient over time can document T cell reconstitution after HCT or increased TRECs following institution of anti-retroviral therapy in patients with HIV-AIDS.
In contrast to its shortcomings as a clinical test, TREC determination, of all the approaches considered to date, is the only one that has proven clinical utility for SCID newborn screening. In 2005, Chan and Puck published the first method for DBS screening for SCID using TRECs as the analyte.50
DNA was extracted from DBS and used for quantitative PCR (qPCR) for TRECs. A β-actin gene segment could be amplified from the same DNA as a control to distinguish samples with low TRECs due to lack of naïve T cells vs. samples with poor DNA yield or quality. As shown in , anonymous DBS samples obtained for routine newborn screening had a mean of 1000 TRECs per punch in the assay as performed in the Puck laboratory. Occasional DBS failed to amplify TREC DNA adequately; such samples would have repeat determinations made and β-actin gene amplification performed, and if both TREC and β-actin PCR failed, as shown in the lower right of the graph in , a new blood spot would be needed from the infant. The initial publication of Chan and Puck had 1.4% of samples with failed PCR, an unacceptably high rate for large scale screening; however, subsequent refinements reduced the false positive rate more than 10-fold (discussed below). Eleven states routinely obtain a second heelstick from all infants at around two weeks of age, making a repeat sample readily available if needed.46
Figure 2 TREC (○) and actin (×) copy number measured in DNA isolated from (A) 300 anonymous dried blood spots (DBS) obtained in newborn nurseries, showing one sample, far right, with failure of DNA amplification; and (B) 20 newborn nursery DBS (more ...)
From states such as California that archive residual dried blood spots from all infants for appropriate research and quality control purposes, we were able to obtain the original nursery DBS samples from 20 infants who were eventually diagnosed with SCID of diverse genotypes. As shown in , TRECs were uniformly undetectable in these samples even though positive amplification of the genomic β actin DNA was confirmed in each case.50,51
Morinishi et al. obtained similar results from newborn blood spots recovered from 5 SCID infants in Japan.52
Advantages of TRECs as a newborn screening analyte include ability to use dried blood spots, low cost, high throughput, and high sensitivity—that is, avoidance of false negative results from infants with SCID who have high numbers of B lymphocytes, maternal T cell engraftment52,53
or oligoclonally expanded T cells. Furthermore, additional conditions in which T cell numbers are low, such as complete DiGeorge syndrome, would also be predicted to be found. Complete DiGeorge syndrome is potentially treatable by thymus transplantation.54
The TREC method was first adapted to high throughput use in a public health setting by the Wisconsin State Newborn Screening Program, which started a state-wide pilot SCID screening program in 2008.55,56
California, New York and other states have followed. In addition to maintaining high sensitivity for detecting SCID cases, TREC test specificity has been excellent with automated methods that have reduced the number of DNA amplification failures to 0.1% or lower. This is as good or better than the false positive rates of many tests currently in use for newborn screening for other conditions.