Genomics, transcriptomics, proteomics, and numbers

Archives of Pathology & Laboratory Medicine, Sep 2003 by Kiechle, Frederick L, Holland-Staley, Carol A

Objective.-To review the advances in clinically useful molecular biologic techniques and to identify their applications in clinical practice, as presented at the 11th Annual William Beaumont Hospital DNA Symposium.

Data Sources.-The 8 manuscripts submitted were reviewed, and their major findings were compared with literature on the same or related topics.

Study Selection.-Manuscripts address the use of molecular techniques in microbiology to evaluate infectious disease and epidemiology; molecular microbiology methods, including rapid-cycle real-time polymerase chain reaction; peroxisome proliferator-activated receptor [gamma] as a potential therapeutic target in inflammatory bowel disease or colon cancer; the effect of nonapoptotic doses of the bisbenizamide dye Hoechst 33342 on luciferase expression in plasmid-transfected BC3H-1 myocytes; the routine use of cystic fibrosis screening and its challenges; and the use of flow cytometry and/or chromosomal translocation in the diagnostic evaluation of hematopoietic malignancies.

Data Synthesis.-Three current issues related to the use of molecular tests in clinical laboratories are (1) the restriction on introducing new tests secondary to existing patents or licenses; (2) the preanalytic variables for the different specimen types currently in use, including whole blood, plasma, serum, fresh or frozen tissues, and free-circulating DNA; and (3) the interpretation of studies evaluating the association of complex diseases with a single mutation or single-nucleotide polymorphism. Molecular methods have had a major impact on infectious disease through the rapid identification of organisms, the evaluation of outbreaks, and the characterization of drug resistance when compared with standard culture techniques. The activation of peroxisome proliferator-activated receptor [gamma] stimulated by thiazolidinedione is useful in the treatment of type II diabetes mellitus and may have value in preventing inflammatory bowel disease or colon cancer. Hoechst 33342 binding to adenine-thymine-rich regions in the minor groove of DNA is a fluorescent stain for DNA and initiates apoptosis at >10 [mu]g/mL. Lower doses of Hoechst 33342 promote luciferase expression by a mechanism that may involve binding to cryptic promoters facilitated by dye-associated misalignment of the tertiary structure of DNA. The routine use of cystic fibrosis screening is complicated by the more than 1000 mutations associated with the disease. The use of 4-color flow cytometry and the detection of chromosomal translocation are both invaluable aids in establishing the diagnosis of lymphoid or myeloid hematopoietic malignancies.

Conclusions.-The current postgenomic era will continue to emphasize the use of microarrays and database software for genomic, transcriptomic, and proteomic screening in the search for useful clinical assays. The number of molecular pathologic techniques will expand as additional disease-associated mutations are defined.

This issue of the Archives of Pathology & Laboratory Medicine features 8 papers presented at the 11th Annual William Beaumont Hospital DNA Symposium, titled "DNA Technology in the Clinical Laboratory," held from September 12 to 14, 2002, in Troy, Mich. The meeting was held on the eve of the 50-year anniversary of the discovery of the structure of DNA by J. D. Watson and F. H. C. Crick published in Nature, April 25, 1953.1,2 Research and development in this postgenomic era continue to focus on identifying the critical messenger RNAs (transcriptomics) or the proteins (proteomics) and the intracellular signaling reactions required to initiate specific biochemical events.3-9 Numbers continue to be refined to define the impact of the genome on phenotypic expression. For example, there are 40 000 to 70 000 genes in the human genome encoding 250 000 to 1 million proteins whose synthesis is regulated by 10000 transcription factors.10,11 Intracellular signal transduction pathways define the phenotype.12,13 The complexity is obvious, and the opportunities for multiple interrelated pathways suggest that a gene regulatory network or circuit is the best way of presenting intracellular metabolic regulation.12-14 For example, there are at least 30 members of the tumor necrosis factor family of receptors, and 8 contain the death domain in their cytosolic tail, which may initiate programmed cell death (apoptosis) by activating one or more of the 11 cysteine aspartyl-specific proteases (caspases).15 In humans, there are 29 ligands with a transforming growth factor B binding domain.16 There are 7 transcription factors called signal transducers and activators of transcription (STATs) in the human genome.16 They are cytoplasmic proteins that dimerize after tyrosine phosphorylation and translocate into the nucleus to function as a transcription factor(s). For example, STAT 5 is a latent cytoplasmic transcription factor that is activated by the erythropoietin receptor as well as many other hematopoietic and nonhematopoietic cytokine receptors17 and that promotes adipogenesis in nonprecursor cells.18

STAT 1 and STAT 3 are subtypes of the STAT family and share a DNA-binding domain and phosphorylation sites for Janus kinase and mitogen-activated protein kinase. Both of these STATs form heterodimers. STAT 1 and STAT 3 have opposing roles in the apoptotic process. STAT 1 induces apoptosis by activating the promoter of the proapoptotic caspase 1, and the overexpression of STAT 3 antagonizes the apoptosis-promoting effects of STAT 1.19 The inhibition of STAT 3 signaling in primary effusion lymphoma cells by the transduction of dominant-negative STAT 3 or the pharmacologic inhibition of Janus kinase 2 with tyrphostin AG490 induces apoptosis and decreases survivin expression.20 Survivin is 1 of 8 members of the inhibitor of apoptosis protein family.15,21 The numbers of factors that promote or inhibit a process are documented as well as the cross talk between apparently unrelated, biochemical processes. The numbers on paper seem to increase exponentially in direct relationship to the apparent complexity of the problem to be unraveled.