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                                           BIOINFORMATICS COLLOQUIUM
					       College of Science
                                            George Mason University
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                      Making Rare Diseases More Complex with Genomics: Eye on Childhood Cancer					


						   James Taylor

					  National Institutes of Health



Abstract:

The initial assembly of human genomic sequence led to anticipation for rapid advances 
in understanding the genetic basis of human diseases.  Efforts are now focused on 
whole-genome genotyping and DNA sequencing to characterize relationships between genotype 
and phenotype in complex or multigenic human diseases like diabetes and cancer.  A major 
challenge for these studies is the ability to distinguish between neutral and functional 
genetic variants and to understand how functional mutations contribute to disease.  Our 
laboratory's mission is to map and characterize medically important polymorphisms and 
mutations in rare human diseases using bioinformatic and laboratory based methodologies.  
In one example, we have examined childhood cancers which are a collection of exceptionally 
rare diseases where their rarity severely limits suitable numbers of tumor samples 
available for genomic studies.  Rhabdomyosarcoma (RMS) is a childhood cancer originating 
from skeletal muscle that affects approximately 250 children each year in the US. Patient 
survival is poor in the presence of metastatic disease, and few determinants that regulate 
metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly 
expressed in RMS tissue suggesting a role in tumorigenesis, although its functional importance 
has not been elucidated. Examination of existing expression databases demonstrated that higher 
FGFR4 expression in RMS tumors is associated with advanced-stage cancer and poor survival, 
while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung 
metastases when the cells were transplanted into mice. This led us to hypothesize that FGFR4 
activation though either overexpression or mutation might contribute to disease progression 
and metastasis.  Targeted gene sequencing identified FGFR4 tyrosine kinase (TK) domain mutations 
among 7% of primary human RMS tumors. Mutations occurring at two codons in the FGFR4 TK domain 
were predicted to promote receptor phosphorylation using bioinformatic algorithms and protein 
structural modeling.  Functionally, FGFR4 mutants K535 and E550 increased autophosphorylation, 
STAT3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS 
cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic 
phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were 
substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor 
than the control cell lines expressing the empty vector or wild-type FGFR4. Together, these 
data demonstrate that mutationally activated FGFR4 functions as an oncogene in RMS and these 
are believed to be the first known mutations in a receptor tyrosine kinase in this tumor. 
These findings support the potential therapeutic targeting of FGFR4 in RMS.  Overall, this 
study also demonstrates the importance of integrating genetic, bioinformatic and archived 
databases for the study of rare human diseases.