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Published: March 2013Print Record of Viewing
Once identified, newborns with SCID can receive life-sustaining therapy. Delays in identification of affected infants rapidly reduce the success of such treatments. In Part 1 of this 2-part presentation, Dr. Abraham discusses the diagnostic path, use of appropriate laboratory tests and test interpretation tools, and potential treatments for this deadly condition. In Part 2, Dr. Abraham discusses the appropriate use of tier 1, 2, and 3 follow-up testing for abnormal newborn screening for SCID/T cell lymphopenia. Due to the complexity of this information, both recordings are now available.
Presenter: Roshini Abraham, PhD
Today’s presentation is going to be on Severe Combined Immunodeficiencies or SCID, and the use of newborn screening to identify infants with SCID or T-cell lymphophenia. The term “Severe Combined Immunodeficiencies or SCID” refers to a group of primary immunodeficiencies (PID), related to genetic defects affecting the immune system. SCID does not refer to a single defect, but rather a heterogeneous group of monogenic disorders. The critical component of the immune system almost universally affected in SCID is the T-cell compartment that performs cellular immune functions. Additionally, other lymphocytic components of the immune system, such as B and/or NK cells may also be affected. Fully penetrant and classic phenotypic manifestations are associated with what is known as “typical forms of SCID.” However, partial loss of function, also called hypomorphic mutations, may be associated with what is known as “leaky SCID,” or Omenn syndrome, if the other characteristic features of Omenn syndrome are also present. This will be discussed later in this presentation. While different reports may vary slightly on exactly how many individual genetic defects are associated with a SCID phenotype, it may be reasonable to include in this list at least 15 to 19 genes, which can present with a severe immunodeficiency. But, this list is by no means complete and new genes are added as they are identified, including one, which is MTHFD1 deficiency, which was reported in a publication as recently as early 2013.
As alluded to in the previous slide, there are several genetic defects that result in a SCID phenotype; however, there is also clinical and immunological heterogeneity, which can pose a challenge to facile classification and diagnosis, especially with leaky SCID or variant SCID (where the molecular defect remains unknown). Despite this confusing heterogeneity, a common feature of all SCID forms is a defect in T-cell numbers (and function in many cases) resulting in a significant susceptibility to infections – be they viral, bacterial, or fungal. Also, these patients often demonstrate a failure to grow normally referred to as failure to thrive (FTT). Due to this profound immunodeficiency, most classic forms of SCID are associated with almost universal mortality by the age of 2 years, if there is not appropriate and timely intervention. Since the majority of these patients appear asymptomatic at birth, and the median age of diagnosis is 4 to 7 months, early diagnostic measures are required before life-threatening complications set in. For this reason, SCID as a clinical entity is well-suited for a population-based screening program such as newborn screening (NBS).
Prior to newborn screening, the reports on the incidence of SCID was variable from report to report and population to population. The result of the initial newborn screening studies suggests that the overall incidence of SCID may be closer to 1:50,000, though this can vary from population to population, with some at higher risk than others. Another strong argument for population-based early, that is newborn, screening for these disorders is the availability of life-saving treatment options for these infants, including hematopoietic cell transplantation; gene therapy, which is available (for some forms of SCID); and enzyme replacement. Early diagnosis, besides being of value to the patient, is also helpful in counseling family members and performing risk assessment, and when appropriate (particularly in certain situations where early treatment can be instituted), prenatal diagnosis. The first clinical case of SCID was identified more than 50 years ago, and the first molecular defect associated with a SCID phenotype was identified in 1972 with ADA (adenosine deaminase) deficiency. It took another 2 decades to identify the next molecular defect associated with X-linked SCID caused by mutations in the IL2RG gene that encodes for the common gamma chain. This latter defect was the one present in the well-known “boy in the bubble” syndrome.
The majority of SCID defects are autosomal recessive except for some forms that are X-linked, as mentioned in the previous slide. In addition to SCID, there are other chromosomal anomalies, such as DiGeorge and CHARGE syndromes, which are associated with autosomal dominant forms of T-cell lymphopenia. In the classic or typical forms of SCID, genetic defects related to T-cell development cause T-cell lymphopenia where patients have an absolute lymphocyte count (ALC ≤500 cells/mcL). The lower limit of normal for newborns and infants is provided in the slide. Since lymphocyte counts in adults are lower than that in children, lymphopenia in adults is typically classified as <1000 cells/mcL. In “leaky” forms of SCID, there may not be an appreciable lymphopenia or, if present, it may be very modest or in some cases there could be lymphocytosis (as seen in some Omenn syndrome patients). Additional analysis often reveals that these T cells, which are present, are abnormal in terms of a very restricted repertoire, ie: they are oligoclonal T cells, most of these T cells express a memory marker (CD45RO) with very few, if any, being naïve expressing CD45RA+ marker. Further, these T cells have abnormal function (as measured by proliferation to mitogen) and low-to-absent TREC (T-cell receptor excision circles), which will be discussed in detail toward the end of the presentation. When T cells are present in an infant suspected of having SCID, it is important to rule-out maternal engraftment and ensure that these T cells are autologous. This can be done in male infants by XX/XY FISH analysis and, in female infants, with short-tandem repeats or STR analysis.
It may be reasonable to pause here for a moment to clarify the term “Omenn syndrome.” Omenn syndrome (OS) refers to a SCID phenotype caused by hypomorphic or partial loss of function mutations in genes, which are typically associated with a classic SCID phenotype. Additionally, these patients may have erythrodermia or skin rash, eosinophilia, adenopathy, hepatosplenomegaly, elevated IgE, oligoclonal autologous T cells with normal numbers or expanded. If some of these features are present, it may be regarded as an atypical OS. While, hypomorphic defects in approximately 7 or so genes have been described thus far, it is possible that there may be additional defects in yet undefined genes associated with this phenotype.
Before continuing our discussion on SCID, let us consider for a moment the role of T cells in cellular immunity. Antibodies, which compose the humoral arm of adaptive immunity, can only access pathogens in blood or extracellular space. Intracellular pathogens, which can include certain bacteria, all viruses, and certain parasites, replicate within cells and remain inaccessible to antibodies. To tackle this component of host defense, T and NK cells act as the primary effector cells. There are different types of T cells, the majority of circulating T cell express the alpha-beta T-cell receptors, while a minority express the gamma-delta receptors. T cells can be divided into subsets based on cell-surface markers. The 2 main subsets of T cells are CD4 T cells (helper) and CD8 T cells (cytotoxic). CD4 T cells can be further subdivided into more granular subsets based on either cytokine production and/or function.
This slide describes the incidence of SCID or SCID-like disease associated with some of the more common monogenic defects. Of note, 3 genes – the common gamma chain, ADA gene, and IL-7R alpha gene, account for close to three-fourths of all SCID defects described thus far. Reticular dysgenesis and cartilage hair hypoplasia are distinct clinical entities in their own right; however, they have sometimes been associated with SCID because of some aspects of their immunological and clinical phenotype.
Besides some of the more commonly described genetic defects associated with SCID, other genetic defects may be associated with T-cell lymphopenia, and some of these have been described from newborn screening analysis, while others are associated with a non-SCID combined immunodeficiency and have T-cell lymphopenia to varying extent. We will be developing a Next Generation sequencing panel for these genes and other primary immunodeficiency genes over the course of this year and the next.
Early diagnosis of SCID is essential because long-term outcomes are influenced by the time at which hematopoietic cell rescue is performed. In a landmark study by Dr. Rebecca Buckley of Duke University, which looked at survival rates posthematopoietic cell transplant (HCT) in infants transplanted before or after 3.5 months of age, 96% of 46 infants transplanted early (that is before 3.5 months of age), survived for more than 24 years posttransplant, while only 66% of 113 infants transplanted at a later date (that is after 3.5 months of age), survived for more than 24 years posttransplant (70% survived for the first 8 years posttransplant). This study emphasizes the need for early diagnosis, which alone can facilitate early intervention.
Therefore, the diagnosis of SCID is all about timing and, of course, making the right diagnosis. It fulfills all the criteria for newborn screening in that it is a lethal disorder for which treatment exists if intervention is offered early enough, and there is a test available for population-based screening (the TREC assay, which will be discussed later in this presentation). However, it is important to note that low T cells can be seen in other conditions apart from SCID, as alluded to in previous slides. Conversely, T-cell numbers can be elevated in the context of leaky SCID, or Omenn syndrome, or maternal engraftment. Typically, in a newborn or infant, low-to-absent TREC copies are associated with T-cell lymphopenia due to decreased or absent thymic output. Once diagnosis has been established, treatment can be instituted, which would lead to improved outcomes as well as enhanced quality of life.
In a recent study comparing the benefits and risks of early vs late diagnosis of SCID, of a total of 158 infants studied, 138 were not tested at birth, and only 58% of these survived, while 42% died. On the other hand, of the 20 infants that were tested at birth, 85% survived and only 15% died. Among the 61 deaths in this cohort of infants, 51% were diagnosed and treated, while 20% were diagnosed and not treated, and another third (29%) were not diagnosed.
When trying to estimate the prevalence of SCID in the United States, or the prevalence of all PID in the United States, it appears that primary immune deficiency is likely to be more common than previously estimated based on a screening phone-based population survey conducted by Dr. Buckley and her group. But all such surveys have their short-comings when estimating true population prevalence and incidence, and only population-based screening of newborns can effectively determine the incidence and prevalence of these diseases.
Since SCID as a broad clinical entity is a lethal disease, it is most reasonable to select such a disorder to be included in newborn screening and 2 recent publications (1 from the United States and 1 from the United Kingdom) make a strong and cogent argument on implementing newborn screening and its value and the process of setting up a population-based screening approach.
The newborn screening process consists of certain universal elements, which include testing of the specific analyte on dried blood spots (DBS) and following up abnormal screening results with additional testing and clinical evaluation to facilitate appropriate intervention and management. Newborn screening, of necessity, mandates that diagnostic testing be available to workup infants identified by screening. Often, the management of these patients is complex and requires multispecialty care. Another advantage of such a population-based screening approach is that it is self-instructive and the results gathered from screening serve to constantly improve the process of screening, but also follow-up and interpretation of results.
The first state to start a statewide process for newborn screening for SCID was Wisconsin in 2008. They developed an assay for quantitative assessment of TREC (T-cell receptor excision circles) on dried blood spots. Using this approach, they were able to identify infants with T-cell lymphopenia.
In January 2010, the Federal Advisory Committee recommended addition of SCID to the universal newborn screening panel, which was a milestone in the history of primary immune deficiencies. Prior to initiation of newborn screening, the incidence was estimated at 1:100,000, though some reports indicated a higher incidence at 1:40,000. The NBS program will help define the true incidence of SCID in the United States, and this may vary by population.
In a pilot program for new born screening for SCID, funded by the NIH and CDC, 7 states including Wisconsin participated and 14 cases were identified between all these states for a million infants tested till April 2011. Since then, additional cases have been described in these states. This data was kindly provided by Dr. Amy Brower of the Newborn Screening Translational Research Network (NBSTRN) and the American College of Medical Genetics (ACMG).
This slide shows implementation of NBS screening for SCID across the United States as of January 2013. 12 states have implemented screening statewide, while 2 states are performing NBS in selected populations. An additional 9 states have pilot programs or statewide screening slated for implementation in 2013. Newborn screening has not yet been implemented for SCID in 28 states.
The newborn screening approach for SCID involves testing of the dried blood spot for a T-cell-specific marker called TREC previously defined (T-cell receptor excision circles) using a molecular method, which is quantitative real-time PCR. If the result of the screen is normal, there is no additional follow-up. If the initial result is abnormal, additional evaluation is initiated based on individual algorithms implemented by state health laboratories. When the state lab ensures that a given sample is abnormal for TREC, follow-up testing (called Tier 1 in some states) is initiated, again as per state-specific protocols.
To understand why the TREC assay is used for identification of T-cell numbers in the newborn screening assay, it is essential to have a basic understanding of T-cell development in the thymus. T cells develop from common lymphoid progenitors (which in turn are derived from hematopoietic stem cell precursors) and these T cells undergo maturation within the thymus (an organ located in the anterior chest cavity near the heart). There are several stages of T-cell development within the thymus and this is a balanced and complex process. However, one of the key events that define T-cell development within the thymus is the process of producing a T-cell receptor (TCR) through somatic recombination and rearrangement of various gene segments (known as V(D)J gene rearrangement). During this process of TCR rearrangement, a DNA product known as TREC is produced. TREC, therefore, stands as a marker for naïve (new) T-cell generation in the thymus and subsequent export into peripheral circulation.
There are many different TRECs generated in the thymus during T-cell receptor rearrangement, depending on which T-cell receptor is being rearranged. Since TREC is not integrated into chromosomal DNA, it is diluted by cell division. Therefore, the TREC considered most representative of thymic output is the dRECjJa signal joint (sj) TREC, and it is this TREC that is measured in the newborn screening assay as well as other clinical lab assays that measure TREC.
The thymus actively produces T cells from birth till puberty. After puberty, thymic activity does not cease completely but, as thymic epithelial tissue is replaced by adipose tissue, there is a proportionate decrease in naïve T-cell production by the thymus. However, there is detectable naïve T cells and TREC-positive T cells into the sixth to seventh decade of life, albeit much less than what would be seen in a neonate or infant.
Thymic output of naïve T cells with diverse rearranged T-cell receptors (TCRs) is essential to maintaining T-cell immune competence. As previously mentioned, normal thymic function in infants and prepubertal children results in a diverse repertoire of T cells, which are shown in the illustration on the left. However, postpuberty and into adulthood, T-cell homeostasis is achieved by a balance between thymic output and peripheral expansion of preexisting T cells. If thymic output is diminished or lost prematurely or aberrantly, then T-cell receptor diversity is extremely constrained (the representation on the right), which would result in decreased immune competence and oligoclonality of the T-cell receptor repertoire. As mentioned earlier, this can be seen in patients with Omenn syndrome, where there is expansion of a limited number of T cells, it can also be seen in “leaky” SCID.
To facilitate implementation of newborn screening-SCID and T-cell lymphopenia across the country and internationally, the Clinical Laboratory Standards Institute (CLSI) is publishing a new guidelines document on the measurement of TREC in dried blood spots. This document is expected to be published within a month or so and would be available to any laboratory or individual interested in understanding, and implementing, newborn screening for SCID and T-cell lymphopenia in greater detail.
The next 2 slides describe the classification used to define SCID and other categories of T-cell lymphopenia that are identified by newborn screening. This classification is shared by the Primary Immunodeficiency Transplant Consortium – PIDTC, the previously mentioned CLSI document, and the R4S database, which is a database collecting newborn screening data for SCID/T-cell lymphopenia. In this classification, there are 6 categories, including classic or typical SCID, leaky SCID, and Omenn syndrome, variant SCID,
other non-SCID syndromes, which have an associated T-cell lymphopenia, other nongenetic causes of T-cell lymphopenia, and the lymphopenia associated with premature births.
As mentioned before, the R4S database is a data repository supported by the Newborn Screening Translational Network (NBSTRN), among others, and is meant to facilitate state labs performing newborn screening for SCID to enter their data for positive cases so that the cumulative data may be analyzed and used mutually to benefit all programs and participants.
No discussion of SCID would be complete without at least a brief mention of precautions to be observed in infants identified as having abnormal TREC by newborn screening, while further workup is in progress. Infants suspected of having SCID or a severe T-cell lymphopenia should be placed in protective isolation and strict care in hygiene should be practiced for these babies. Also, breast feeding should be avoided to prevent CMV transmission until the infant has been tested further. All live vaccines should be avoided and only leukodepleted and irradiated blood products should be used, if a blood transfusion is necessary. Antibiotic prophylaxis may also be used as determined by the infant’s physician.
As discussed earlier in the presentation, the definitive treatment for typical SCID and other appropriate clinical categories is hematopoietic cell transplantation (HCT). In patients with ADA deficiency, enzyme replacement with PEG-ADA can be used until arrangements are made for hematopoietic cell transplantation. Also, some centers in the United States and overseas offer gene therapy for certain types of SCID, including ADA deficiency and X-linked SCID. On identification of an abnormal NBS result for TREC, the care of such infants fall primarily to clinical immunologists, who work with pediatricians and other specialists in providing the best and most expedient care for these critically ill babies.