A synonymous mutation V107V in Factor IX is not silent: possible cause of Hemophilia B

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	FDA/CBER	BIOCHEM/CHEM-23

Authors

• V Simhadri
• N Katagiri
• S Tseng
• R Zichel
• N Edwards
• M Stern
• D Kopelman
• J Muste
• Z Sauna
• A Komar
• C Kimchi-Sarfaty

Abstract

Hemophilia B is characterized by defects in coagulation factor IX (FIX) caused by gene alterations, including a synonymous mutation (reported by Knobe and colleagues (Hemophilia, 2008)). However, the mechanism by which the synonymous mutation can precipitate disease remains elusive. Here we have performed in silico analyses of the synonymous codon substitution (GTG to GTA) of the V107V mutation and observed a change in the structure and stability of mRNA and its codon usage. Interestingly, the rate of synthesis of the mutant protein was slower in an in vitro translation system due to substitution of the frequent GTG to the rare codon GTA (28.12 to 7.2/1000 human codons). FIX stable cell lines were characterized for the levels of mRNA and protein expression and function: no change in mRNA levels was observed, but protein expression and function of the V107V mutant was significantly reduced compared to the wild-type protein. Further analysis by native gels and trypsin digestion experiments suggests conformational differences in the mutant protein. The stability of the mutant protein was also shown to be different than the wild-type. These results indicate that the V107V synonymous mutation in FIX is not silent and may be the cause of mild hemophilia B.

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