Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein.
X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.
Lee
Hee Keun
HK
Department of Biology, Kyungpook National University, Daegu, South Korea.
Song
Mee Hyun
MH
Kang
Myengmo
M
Lee
Jung Tae
JT
Kong
Kyoung-Ah
KA
Choi
Su-Jin
SJ
Lee
Kyu Yup
KY
Venselaar
Hanka
H
Vriend
Gert
G
Lee
Won-Sang
WS
Park
Hong-Joon
HJ
Kwon
Taeg Kyu
TK
Bok
Jinwoong
J
Kim
Un-Kyung
UK
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D003638:Deafness / Q000235:genetics*; D005192:Family Health; D005260:Female; D040181:Genetic Diseases, X-Linked / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D050809:POU Domain Factors / Q000737:chemistry / Q000235:genetics*; D010375:Pedigree; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D050809:POU Domain Factors; C091774:POU3F4 protein, human; D011993:Recombinant Fusion Proteins; D008156:Luciferases