Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism
J. Clin. Invest. Nelly Pitteloud, et al. 117:457 doi:10.1172/JCI29884 [Go to this article.]

Figure 4
A 8-bp intronic NELF deletion results in missplicing of exon 10. (A) Direct DNA sequencing of cloned PCR products of the proband’s genomic DNA revealed a heterozygous 8-bp deletion in intron 9. This deletion is part of a direct 8-bp repeat (tgtggcct) and occurs 14 bp upstream of the exon 10 acceptor (5′) splice site. The lower case part of the sequence indicates introns while the upper case indicates exons. (B) Predicted cDNAs in the exon 8–11 region of the NELF gene. The proband was found to have an 8-bp deletion in intron 9 (black triangle), 14 bp upstream of exon 10. (C) The result of RT-PCR using HEK-293 cell mRNA from cells transfected with WT NELF genomic construct containing exons 8–11 and the mutant NELF construct (8-bp deletion in intron 9). The HEK-293 cells transfected with the WT NELF construct show a normally spliced NELF exon 8–11 RT-PCR product, corresponding to the predicted size of 291 bp. The cells transfected with the mutant NELF construct, however, show an additional band of 257 bp corresponding to the expected size of a transcript lacking exon 10. The RNA from cells transfected with either WT or mutant genomic construct show an additional product of 794 bp (786 bp for the mutant construct), reflecting PCR amplification of the residual plasmid DNA. (D) Colocalization of NELF and GnRH1 in the olfactory epithelial cell line FNC-B4 by immunohistochemistry. Original magnification, ×150.