GENOMIC ANALYSIS
Genomic analysis will focus on gene expression with two approaches:
Gene expression microarrays: Microarrays permit the study and comparison of thousands of genes simultaneously in order to obtain measures of all the expressed genes from blood or tissue samples and then make comparisons with other samples to identify differentially expressed genes. Expressed gene profiles from blood or tissue will be generated from samples collected longitudinally over the course of a clinical trial or in natural history studies for disease staging. With this technology, researchers can study which genes are turned on or off by either disease or treatment and formulate a detailed picture of the genetic profile of a specific disease and its treatment on patient groups or individuals over the course of a clinical trial. Differential gene expression profiles representative of individual disease or treatment groups can then be determined. Individual patient profiles can be generated by comparing and contrasting samples in a longitudinal series over the course of a clinical trial.
Quantitative gene expression: Validation of expressed gene markers requires the use of a quantitative method in order to determine a more precise level of gene expression not possible from microarrays. This quantitation is often the second step in developing a validated expressed gene marker for clinical monitoring or diagnostics. However, single quantitative real-time PCR reactions have proven to be quite laborious and expensive, thereby limiting the number of genes able to be examined simultaneously. Recent advances in the multiplexing of real-time PCR reactions are circumventing this problem, allowing quantitative real-time PCR to be used to examine hundreds of genes at once. Unlike traditional approaches, ITI will employ newer technologies that will examine precise levels of expression of hundreds of genes at once, providing a more in-depth genetic profile of disease progression and treatment effects.
ITI will utilize these methodologies for:
- Quantitation of mRNA transcripts of hundreds of genes
- Profiling of microRNA
- Epigenetics-such as DNA methylation
Immunological Disease Screening: In January 2009, the Institute launched a major initiative, together with ITI Board member Dr. Jennifer Puck and in collaboration with the biotechnology company Sequenom, Inc., to establish widespread newborn screening to identify infants with Severe Combine Immune Deficiency (SCID). SCID, an illness in which the infant fails to develop a normal immune system, is the most severe of the Primary Immune Deficiency diseases. SCID babies can be infected by a wide range of viruses, bacteria and fungi that are normally controlled by a healthy baby's immune system. If undetected and untreated, SCID typically leads to death before the baby's first or second birthday.
Bone marrow or stem cell transplantation and enzyme replacement have changed this previously fatal disease to a treatable one. Gene therapy is also a promising treatment. Importantly, the sooner a child is diagnosed, the sooner treatment can begin and the more likely it is to be effective. For example, SCID infants identified by a prior family history and treated early in life have better survival, less morbidity and significantly lower treatment costs than those identified only after serious infections. Recent research also confirms that bone marrow transplants in the first three months of life work better than transplants at a later age. So it is critical to identify affected children immediately after birth. Unfortunately, most infants with SCID today are not identified in the pre-infectious period.
A universal newborn screening program could remedy this problem. While SCID is considered rare (estimated at one in every 50,000-100,000 newborns) there have been no screening programs to evaluate the true incidence. Experts suspect many more children might have SCID and be dying of infections without being diagnosed.
Since SCID babies lack overt clinical symptoms for some time after birth, a molecular test is necessary for early post-natal diagnosis. There are at least 14 known SCID genes, some of which are listed below, which affect lymphocyte development. The most common form, caused by mutations in the X-linked common cytokine receptor gamma chain, was identified in Dr Puck’s lab and accounts for about half of SCID cases.
| Some of The Known Forms of SCID: |
Gene |
Lymphocyte Phenotype |
| |
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|
X-Linked |
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|
Common gamma chain gene mutations |
IL2R-gamma |
T(-) B(+) NK(-) |
|
|
|
Autosomal Recessive |
|
|
Jak3 gene mutations |
JAK3 |
T(-) B(+) NK(-) |
ADA gene mutations |
ADA |
T(-) B(-) NK(-) |
IL-7R alpha-chain mutations |
IL7R alpha |
T(-) B(+) NK(+) |
CD3 delta or epsilon mutations |
CD3 delta or epsilon |
T(-) B(+) NK(+) |
RAG1/RAG2 mutations |
RAG1/RAG2 |
T(-) B(-) NK(+) |
Artemis gene mutations |
ARTEMIS |
T(-) B(-) NK(+) |
CD45 gene mutations |
CD45 |
T(-) B(+) NK(+) |
The hallmark phenotype of all forms of SCID is the lack of T cells, one of the essential types of white blood cells that make up the immune system, while some also lack B cells and NK cells. Without a sufficient number of normal T cells, the immune system doesn't work, just as when the AIDS virus wipes out the same population of immune cells. The diversity of the genetic defects that can produce the SCID phenotype, both in terms of the number of genes and mutations within the genes, makes any universal diagnosis of SCID by germ line genetic analysis difficult.
In this regard, Dr Puck has developed a new laboratory method that rapidly identifies newborns with all T cell deficient forms of SCID. The new test can use the same dried blood samples already collected from newborns (such ‘Guthrie’ blood spot cards are currently collected in all U.S. states to test newborns for inherited diseases such as PKU and hypothyroidism), and would provide the first accurate, high-throughput screen for immune deficiencies. Prior efforts to identify this disorder by counting white blood cells in newborns proved expensive and insufficiently sensitive or specific.
The newly developed screening tool exploits a detailed understanding of the normal maturation of T cells. During normal development, an individual T cell rearranges the gene that encodes the T cell antigen receptor (TCR) on the surface of the cell. The antigen receptor allows the T cell to identify an infectious agent and launch a defensive attack to kill the invader.
While rearranging the receptor gene, the maturing T cell produces as a byproduct a DNA circle within the cell, called a T cell Receptor Excision Circle (TREC). These circles are stable, although they become diluted as T cells proliferate. Quantitative measurement of TRECs would enable diagnosis of all genetic forms of SCID.
Dr. Puck’s assay employs a quantitative PCR reaction across the delta-deletion TREC joint formed by the genomic rearrangement. Quantitating TRECs indicates the number of recently formed T cells in the peripheral blood and is a measure of immune competence.
Chan and Puck have developed a method to extract DNA from Guthrie dried blood spot cards and quantitatively measure TRECs with a real-time PCR (Taqman) assay (J Allergy Clin Immunol 115:391-398, 2005). Using quantitative PCR of TRECs to measure the number of these circles within a blood sample, Dr. Puck's group has been able to differentiate normal infants from those with SCID. In dried blood samples from healthy babies, the team was able to detect an average of 1,000 TRECs; children with SCID had 30 or fewer.
ITI has committed to working in collaboration with Dr. Puck and Sequenom to optimize the TREC assay for newborn screening, utilizing the real-time PCR assay and adapting the assay to the Sequenom MassArrayÒ platform as a quantitative, competitive end-point PCR. Adapting this assay to the Sequenom platform has potential advantages for the overall throughput and cost-effectiveness needed to support newborn screening.
Consensus has emerged from multiple key constituencies, including newborn screening programs, the pediatric immunology community, pediatric transplant centers, and federal (NIH, CDC), state, and nongovernmental agencies, that SCID meets the profile of a disorder of high priority for population-based newborn screening (Curr. Opin. Allergy Clin. Immunol. 7:522-527. 2007). Namely, 1) most affected infants are not brought to medical attention until they develop serious infectious complications, 2) SCID is fatal if untreated, 3) effective treatment with allogeneic bone marrow or hematopoietic stem cell transplantation is widely established, and 4) the best outcome for SCID, as with many other conditions for which newborn screening is now performed, is achieved if hematopoietic stem cell or bone marrow transplantation is performed in the first months of life, ideally before clinical presentation with infections and failure to thrive. Thus, a consortium of physicians from throughout the country – called the SCID Newborn Screening Working Group –is now moving toward establishing universal screening of newborns for SCID.
ITI envisions this initiative for universal SCID newborn screening as a major opportunity to generate important new knowledge about human immune disorders as well as to provide better care of patients for public benefit.
