A look into the point mutation of c-kit that leads to mastocytosis
Mastocytosis is a disease in which excessive amounts of mast cells build up in bone marrow and skin lesions. It has been documented as far back as 1869 in the form of a cutaneous disease accompanied by lesions1. The growth of mast cells is controlled by stem cell factor. This process goes haywire when a point mutation occurs in the proto-oncogene c-kit, specifically an A-T substitution that causes Asp816 to become Val3. This proto-oncogene codes for a transmembrane tyrosine kinase receptor, which helps mediate the growth and proliferation of these cells3. Other immune cells, such as T cells and B cells carry this gene but do not express it on their surface, making them less prone to the disease even if they carry the mutation2. Large advances in understanding the disease and the mutation that causes it have been made in the last twenty years.
Valent1 et al proposed a new classification system for different forms of mastocytosis due to modern advances in understanding different forms of the disease. They note that patients can shift from one form of mastocytosis to another, each with rather different symptoms, even if they are controlled by the same process.
This paper while possibly helpful at the doctor and patient level, does little to address the reasons and mechanics behind the mutation of c-kit and development of mastocytosis. While they attempt to sort varying forms of the disease based on individual patient cases, the paper seems to go in circles and accomplish nothing that can be understood outside the medical community. The differences pointed out between different cases seem trivial, especially when they are all linked back to the c-kit mutation.
Akin2 et al’s research was meant to find the how the Asp816Val mutation was spread amongst hematopoietic cell lines as well as cells with differentiation markers for myelmonocytic cells such as T and B lymphocytes. They hypothesize that mutations in the proto-oncogene c-kit, is related to the genesis of Mastocytosis and that it may result from the enhanced growth of mast cells and their precursor cells that express surface c-kit and the Asp816Val mutation.
With blood samples from eighteen patients with Mastocytosis or urticaria pigmentosa, the desired cells were separated out using immunomagnetic beads. These beads are coated with specific antibodies according to the desired cells. Once the desired cells were isolated, they were prepared for RNA extraction, as the Asp816Val mutation shows in the RNA. Using Moloney murine leukemia virus reverse transcriptase, the RNA was transcribed to cDNA. This DNA in turn was used in a PCR process. The DNA was then cut with Hae III and Hinf I restriction enzymes and run though a polyacrylamide gel and ethidium bromide staining. The bands in the gel were then measured for fluorescent intensity. Peripheral blood was sorted and analyzed with a flow cytometer.
The mutation was detectable in white blood cells, B lymphocytes in one patient, and other monocytes and myeloid cells in another patient. They determined that it was the use of the specific isolation of certain cell lineages that allowed them to track the mutation. The mutation was detectable in the peripheral blood of 4 of the patients. Expression of the mutation was found in bone marrow cells and their lineages. There was concern that the PCR signal would be false from imperfections from the isolation technique with the immunomagnetic beads, but it was shown that a mutated PCR signal was not detectable under their conditions. The cytometric data met expectations by showing there is no detectable c-kit membrane expression in peripheral blood cells.
Nagata3 et al, working in 1995, hypothesized that Mastocytosis was linked to a mutation in c-kit. They noted that stem cells and mast cells express c-kit and that a mutation in c-kit might result in the disease. One of one patient with Mastocytosis had the corresponding mutation in genomic DNA. Finding the point mutation that causes the Asp816Val would provide insight into the pathogenesis of the disease.
Blood samples were obtained from eight patients. Using RNA extraction techniques and Polymerase Chain Reaction, in fact, the same reverse transcriptase from Moloney murine leukemia virus as Atkin et al used, were employed to isolate the target DNA. To check for polymorphisms in the cDNA, single-strand conformation polymorphism (SSCP) was performed on the PCR products, any polymorphisms found were sequenced. These sequences were purified via agarose gel electrophoresis. These purified samples were then amplified and sequenced. The restriction enzyme Hinf I was used to find spots where A had been substituted with a T, which creates a restriction site not found in the normal DNA and therefore leads to different bands in a polyacrylamide electrophoresis.
The experiment showed a nucleotide sequence change in six patients. The A-T substitution was confirmed by the Hinf I digestion of PCR products. In the four c-kit-mutation positive patients, the bone marrow showed proliferative abnormalities.
The experiment showed that four patients with Mastocytosis and an associated blood disorder carried the A-T substitution. The overall data did suggest that the Asp816Val point mutation was responsible for the pathogenesis of mastocytosis. It was found that the majority of cells in blood expressing c-kit are CD34+ cells, which are progenitors of mast cells. Although the researchers considered it likely that all descendents of these cells would express the mutation, they claimed it was not clear at the time. The researchers determined that further screening of the patients and follow-ups were necessary to gain a better understanding of c-kit’s relation to mastocytosis.
In summary, mastocytosis is an uncontrolled proliferation of mast cells cause by a point substitution mutation of the c-kit proto-oncogene causing an Aspartic Acid to become a Valine. The various researchers above sought out to explain why mastocytosis occurs at the genetic and cellular levels. They have pinpointed the location of the genetic error and how it affects the mutated cells.
Hopefully with this knowledge, new treatments for the disease can be developed at the genetic level and be detected sooner than in the past. Certainly more research needs to be done, but understanding how some cell lines start uncontrolled reproduction can lead to understanding other forms of uncontrolled cell division.
1. Valent, Peter, Hans P. Horny, Luis Escribano, B. Jack Longley, Chin Y. Li, Lawrence B. Schwatz, Gianni Marone, Rosa Nunez, Cem Akin, Karl Sotlar, Wolfgang R. Sperr, Klaus Wolff, Richard D. Brunning, Reza M. Parwaresch, K. Frank Austen, Karl Lennert, Dean D. Metcalfe, James W. Vardiman, and John M. Bennett. "Diagnostic Criteria and Classifcation of Mastocytosis: a Consensus Proposal." Leukemia Research 25 (2001): 603-25.Www.elsevier.com/locate/leukres. Web. 31 Mar. 2011.
2. Akin, Cem, Arnold S. Kirshenbaum, Tekli Semere, Alexandra S. Worobec, Linda M. Scott, and Dean D. Metcalfe. "Analysis of the Surface Expression of C-kit and Occurrence of the C-kit Asp816Val Activating Mutation in T Cells, B Cells, and Myelomonocytic Cells in Patients with Mastocytosis." Experimental Hematology 28 (2000): 140-47. Elsevier. Web. 31 Mar. 2011. <http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VP8-3YKKBN0-3&_user=3554035&_coverDate=02%2F29%2F2000&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000060848&_version=1&_urlVersion=0&_userid=3554035&md5=ba092a3e0dac0dd88ac568da5da3228a&searchtype=a>.
3. Nagata, Hiroshi, Alexandra S. Worobec, Chad K. Oh, Badrul A. Chowdhury, Susan Tannenbaum, Yoshifumi Suzuki, and Dean D. Metcalfe. "Identification of a Point Mutation in the Catalytic Domain of the Protooncogene C-kit in Peripheral Blood Mononuclear Cells of Patients Who Have Mastocytosis with an Associated Hematologic Disorder." Proceedings of the National Academy of Sciences 92.23 (1995): 10560-0564. Web. 29 Mar. 2011.