1. Nonsense mutation of EMX2 is potential causative for uterus didelphysis: first molecular explanation for isolated incomplete müllerian fusion 
Shan Liu, M.D., Ph.D., Xuan Gao, M.D., Yingying Qin, M.D., Ph.D., Wen Liu, M.S., Tao Huang, M.S., Jinlong Ma, M.D., Ph.D., Joe Leigh Simpson, M.D., Ph.D., Zi-Jiang Chen, M.D., Ph.D. 
Fertility and Sterility 
Volume 103, Issue 3, Pages e2 (March 2015)
DOI: /j.fertnstert
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Identification of the transcript variant expressed in human uterus. (A) Schematic diagram of the two transcripts of the EMX2 gene. GenBank NM_ (EMX2-001) has three exons and encodes a protein with 252 amino acids. GenBank NM_ (EMX2-201) lacks an exon in the coding region, encoding a protein with 169 amino acids. Specific primers were designed to distinguish the transcript variant expressed in the human uterus. Arrows indicate the positions of the forward primer (F) and reverse primer (R). The predicted PCR products are 302 bp and 117 bp, respectively. (B) Results of agarose gel electrophoresis show PCR products of 302 bp. Four cDNA samples were collected from myometrial tissue obtained from women undergoing myomectomy.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
The novel nonsense mutation detected. (A) DNA sequence chromatograms obtained by direct sequencing showing the heterozygous G-T substitution in EMX2 gene. The arrow indicates the G-T change in the woman with IMF. (B) Sequence alignment of the EMX2 protein among many species. The arrow indicates the position of the mutation. Asterisks (*) indicate amino acid residues that are conserved in all these species (humans, mice, rats, cows, chicken, zebrafish, and frog).
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Western blot analyses confirm that this novel mutation can cause truncated protein. HEK293FT cells were transfected by empty vector, wild-type EMX2, mutant EMX2, and a 1:1 combination of the wild-type/mutant plasmids. The 52-kD and 32-kD protein bands indicate wild-type and mutant EMX2 constructs, respectively.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
The mRNA levels in transfected HEK293FT cells for genes considered to be involved in MDAs. The graph shows the mRNA levels relative to the actin mRNA values. The values of the empty vector transfected HEK293FT cells are arbitrarily set to 1. Data are shown as the mean values from three independent experiments with error bars indicating the standard deviation. (A) The mRNA levels of WNT4, WNT5A, WNT7A, WNT9B, HOXA9, HOXA10, HOXA11, HOXA13, LHX1, PAX2, HNF1B, and AMH show no statistically significant difference among cells transfected by empty vector, wild-type, or mutant vector. (B) Wild-type EMX2 shows a 4.5-fold induction upon P63 expression compared with the empty vector, whereas mutant EMX2 has no activation effect. In the 1:1 combination transfection group, the P63 mRNA level is decreased more than the wild type alone.
Fertility and Sterility  , e2DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions