The consequences of chromosome imbalance: using a trisomic human embryonic stem cell line to determine the primary defects in individuals trisomic for chromosome 13 (Patau syndrome)
Over 50% of all human conceptions have too much or little genetic material, often due to the gain or loss of entire chromosomes. The majority of these are spontaneously aborted, but a significant fraction develop to term. Perhaps the most well known is trisomy 21, Downs syndrome, in which the individual bears three rather than the normal two copies of chromosome 21. Aneuploidy results in severe developmental abnormalities including mental retardation and severe physiological abnormalities. In spite of its frequency and biomedical relevance, we known little about the primary causes of the developmental defects associated with aneuploidy. Unlike Mendelian inherited disease genes, aneuploidy is not due to disruptions in gene sequence rather it is due to changes in gene copy number. Until recently, we did not have the tools to determine the primary molecular defects in aneuplid individuals and studies necessary focused on secondary defects. However the development of human embryonic stem cells and more specifically the isolation of a human embryonic stem cell line containing three rather than the normal two copies of chromosome 13 now enables us to explore the primary defects associated with human aneuploidy. In this proposal we plan to use newly developed methods to explore global patterns of gene expression in normal and trisomic lines. In is our hope that these will provide a glimpse of the initial defects associated with human aneuploidy. If the primary defects are due to the misexpression of only one or two genes, this would offer the possibility of developing theurapetic drugs to compensate for the mis-expressed gene(s). In addition, we propose to explore strategies for rapidly establishing normal cell lines from the trisomic human embryonic stem cell line as a first step toward cell replacement therapy. .
Statement of Benefit to California:
This grant focuses on the primary molecular and cellular defects associated with trisomy 13, also known as Patau syndrome. Approximately 1 in 8,000 live births are trisomic for chromosome 13. Trisomy 13 together with trisomy 18 and 21 account for about 2 of every 1,000 live births and therefore classify as rare diseases. Approximately 25 million Americans (and between 2 and 3 million Californians) suffer from over 6,000 rare diseases for which there is no financial incentive for companies to develop drugs. In response, congress enacted the very successful Orphan drug act that has resulted in an over tenfold increase in the number of drugs targeting rare diseases. More recently, congress enacted The Rare Diseases Act of 2002 in order to produce NIH-sponsored centers of excellence for research on rare diseases. Three of these centers are in California(UCSF, UCLA, UCSD). Due to the severe restrictions on federally funded research with human embryonic stem cells, to my knowledge there is no institutional backing for the application of hESC technologies toward the study and treatment of rare diseases. Funding the project proposed in this grant would be the first step towards laying the groundwork for a general strategy for CIRM to apply their resources toward helping the millions of Californians afflicted by rare diseases.
SYNOPSIS: This application proposes to further our understanding of the effects of chromosome trisomy by studying the effect of trisomy 13 on mRNA and protein expression in human ES cells. In Aim 1 the applicant will use microarrays to determine the effect of trisomy 13 on the global pattern of gene expression in human ES cells and some of its differentiated descendants. In Aim 2 the applicant will use two dimensional gel electrophoresis to similarly examine patterns of protein expression. In Aim 3 the applicants propose to use markers of the diploid state, as determined in Aims 1 and 2, to devise sensitive methods for detecting rare diploid revertants of trisomic cells. SIGNIFICANCE AND INNOVATION: Although aneuploidy is an important cause of human birth defects, the primary molecular consequences of trisomy in the early embryo are not understood. The experiments in this proposal are significant because they will offer the first look at the global effects of a specific form of aneuploidy, trisomy 13, on early embryonic cells. An intriguing, and innovative extension of this work is the proposal that it may be possible to identify molecular markers of the diploid state. These markers may be used to screen for rare diploid revertants among trisomic progenitor cells. It is suggested that diploid revertants, for example in hematopoeitic progenitors, may be useful therapeutically. Insight into the mechanism of initiation of aneuploidy would have profound effects on many fields of research, including cancer. STRENGTHS: This is an innovative proposal that has the potential to form the basis for an new, therapeutic use of human stem and progenitor cells. The applicant has identified collaborators to help with many of the technically challenging aspects of this work. WEAKNESSES: There are numerous weaknesses in this proposal: The applicant has no prior experience in essentially any aspect of the proposed work. He has no prior experience with stem cells, in analyzing gene expression by DNA microarrarys, or in studying global patterns of protein expression by two dimensional gel electrophoresis. Little thought has been given to what biological information might emerge from the proposed experiments, other than the trivial idea that the expression of some genes might be increased more than would be expected by the 1.5 fold increase in gene copy number. For example, what are the specific biological abnormalities in trisomy 13 individuals and how might these be expressed at the cellular level? Thus, what specific progenitor cell types might it be useful to examine, and what molecular pathways might be affected? There is considerable doubt that this approach will result in markers that will be sufficiently sensitive and specific to allow isolation of rare diploid revertants of triploid cells Also, not enough thought has been given to the potential therapeutic utility of diploid revertants derived from trisomic stem/progenitor cells. The applicant mentions only one possibility: the use of hematopoietic progenitors to treat leukemia associated with trisomy 21. However, the applicant does not consider the possibility that this is not completely a cell autonomous phenotype. For instance, could the stromal microenvironment be abnormal, and contribute to the increased incidence of leukemia? PI is only 11% committed to the project. It is stated that he will direct and assist in all aspects of the research. This allocation may not be sufficient. Letters of Support are missing. DISCUSSION: This proposal is innovative, but not thought to be feasible. The revertants will be rare and it is doubtful that the methods will allow detection and isolation of diploid revertants. How will diploid revertants be characterized? In addition, any of the three chromosomes could be lost - the trisomic one or one of the original ones, and not clear how this would be determined or what implications would be.