Novel mutations and polymorphisms in the CFTR gene associated with three subtypes of congenital absence of vas deferens  Xiaojian Yang, M.D., Qipeng Sun, M.D., Ping Yuan, M.D., Ph.D., Hao Liang, Ph.D., Xiao Wu, M.D., Luhua Lai, Ph.D., Yan Zhang, M.D., Ph.D.  Fertility and Sterility  Volume 104, Issue 5, Pages 1268-1275.e2 (November 2015) DOI: 10.1016/j.fertnstert.2015.07.1143 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Partial sequencing results of novel mutations in the patients (left) and normal controls (right) for the CFTR gene. (A) Forward sequences in Patient 2. The arrows indicate the homozygous mutation c.4433C>G (p.Thr1478Arg) of exon 27. (B) Forward sequences in Patient 6. The arrows indicate the heterozygous mutation c.3469-3C>A of intron 21. (C) Forward sequences in Patient 13. The arrows indicate the heterozygous mutation c.1357delT (p.Leu453Cysfs*16) of exon 10. (D) Forward sequences in Patient 18. The arrows indicate the heterozygous mutation c.3407C>T (p.Ala1136Val) of exon 21. CBAVD = congenital bilateral absence of the vas deferens; CPAVD = congenital bilateral partial aplasia of the vas deferens; CUAVD = congenital unilateral absence of the vas deferens. Fertility and Sterility 2015 104, 1268-1275.e2DOI: (10.1016/j.fertnstert.2015.07.1143) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Bioinformatics analysis of four novel mutations and structural modeling of CFTR p.Thr1478Arg. (A) Schematic illustration of the motif and domains in CFTR. The wild-type CFTR protein has 1,480 amino acids and contains 2 transmembrane adenosine triphosphate (ATP)-binding cassette domains (ABC transmembrane type-1), 2 ABC transporter nucleotide-binding domains (ABC transporter-like 1), a CFTR regulator domain, and a PDZ-binding motif. The arrows indicate the locations of three novel mutations and the premature stop codon. The mutation c.1357delT (p.Leu453Cysfs*16) occurred in the ABC transporter-like 1 domain and was predicted to delete 1,012 amino acids from the carboxyl terminus of the CFTR protein. The missense mutation c.3407C>T (p.Ala1136Val) was predicted to disrupt the transmembrane ATP-binding cassette (ABC) domain. Another missense mutation, c.4433C>G (p.Thr1478Arg), occurred in the PDZ-binding motif, which mediates interactions with golgi-associated PDZ and coiled-coil motif containing (GOPC) and the SLC4A7, SLC9A3R1/EBP50 complex. (B) Schematic illustration of the normal splicing of exons 21–22 of the wild CFTR protein and the predicted abnormally spliced mutant protein caused by the splicing mutation c.3469-3C>A, which was predicted to delete 151 base pairs from the 5′ end of exon 22. The overall structure of the wild-type (C1) and p.Thr1478Arg mutant (C2) CFTR PDZ-binding domain binding to the CAL PDZ domain is shown as a cartoon. CAL and CFTR are cyan and magenta, respectively. Residues from CAL involved in the interaction with CFTR are displayed as lines, whereas the TRL motif from the CFTR PDZ-binding domain is shown as sticks. A hydrogen bond between Thr1478 and Arg92 is shown as green dashes. The electrostatic potential of the surface of the CAL PDZ domain together with the wild-type (D1) and p.Thr1478Arg mutant (D2) CFTR PDZ-binding domain indicates positively and negatively charged regions blue and red color, respectively. Interacting residues of CFTR are shown as a cartoon and sticks, colored magenta. Structural display and electrostatic potential surface generation were performed with PyMOL (http://www.pymol.org/). Fertility and Sterility 2015 104, 1268-1275.e2DOI: (10.1016/j.fertnstert.2015.07.1143) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions