Unique haplotype in exon 3 of cone opsin mRNA affects splicing of its precursor, leading to congenital color vision defect.
We have analyzed L/M visual pigment gene arrays in 119 Japanese men with protanopia color vision defect and found that five had a normal gene order of L-M. Among the five men, two (identified as A376 and A642) had apparently normal L genes. To clarify their L gene defect, the whole L or M gene from A376 and control subjects was cloned in an expression vector. Total RNA extracted from the transfected HEK293 cells was analyzed by Northern blot and reverse transcription-polymerase chain reaction. The product from the cloned L gene of A376 was smaller than the normal control due to the absence of exon 3. To investigate such exon-skipping at splicing, minigenes of exon 3 accompanying introns 2 and 3 were prepared from A376, A642, and control subjects. The minigenes of A376 (L) and A642 (L) showed the product lacking exon 3 only, while the minigene of normal control N44 (L) showed the product retaining exon 3 only. Exchanging of introns 2 and 3 between the A376 (L) and N44 (L) minigenes showed that the skipping of exon 3 was caused by the exon itself. Seven differences in exon 3 between A376 (L) and N44 (L) were all within already-known polymorphisms as follows: G(151-3), C(153-1), G(155-3), A(171-1), T(171-3), G(178-1) and G(180-1) in A376 (L) and A642 (L), and A(151-3), A(153-1), C(155-3), G(171-1), G(171-3), A(178-1) and T(180-1) in N44 (L). An in vitro mutagenesis experiment with these nucleotides in the minigenes showed that exon 3 was completely skipped at splicing only in the haplotype observed in A376 (L) and A642 (L). These results suggest that complete skipping of exon 3 at splicing, due to the unique haplotype of the exon, causes loss of expression of L-opsin in these men.
Department of Molecular Medical Biochemistry, Shiga University of Medical Science, Seta, Otsu 520-2192, Japan. datt@belle.shiga-med.ac.jp
Hisao
H
Ueyama
Sanae
S
Muraki-Oda
Shinichi
S
Yamade
Shoko
S
Tanabe
Takahiro
T
Yamashita
Yoshinori
Y
Shichida
Hisakazu
H
Ogita
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D044466:Asian People / Q000235:genetics; D003117:Color Vision Defects / Q000235:genetics*; D055358:Cone Opsins / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D057809:HEK293 Cells; D006239:Haplotypes; D006801:Humans; D007564:Japan; D008297:Male; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D012333:RNA, Messenger / Q000235:genetics; D017299:Rod Opsins / Q000235:genetics*
D055358:Cone Opsins; D012333:RNA, Messenger; D017299:Rod Opsins; C095855:long-wavelength opsin