PMC2792980_01
GHR polypyrimidine tract. (A) Schematic representation of the typical eukaryotic intronic nucleotide sequence upstream the intron/exon splice junction (top) and the nucleotide composition of the intronic region upstream GHR exon 8 (bottom). Pyrimidines are shown in uppercase letters; position and length of the polypyrimidine tract (Poly-Y) are also indicated. (B) IVS7-6T>A. Chromatograms showing partial DNA sequences for the patient, his parents and a normal control are presented. The position of the mutation is indicated by the arrows. Y, pyrimidines; n, any nucleotide.
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fig1
In both parents, the same mutation was present in heterozygosity (Fig. 1).
Results for the in vitro splicing assay. The wild-type (Wt) and mutant (Mt) minigenes L1-GHRexon8-L2 and Adml-par were incubated in HeLa nuclear extracts for 60 min at 30  C. Control reaction mixtures were incubated at 0  C. The structures of the pre-mRNA and the mRNA splice products are indicated next to the autoradiogram. The expected size of the splice products is also indicated. Bands corresponding to correct and aberrant mRNA splice products are indicated by the arrows.
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fig2
No band of 277nt corresponding to L1-GHRexon8-L2 was present among the mutant minigene splice products (Fig. 2).
Effects of the GHR T to A nucleotide change on mRNA splicing in HEK293 cells. (A) Radioactive RT-PCR analysis performed with mRNA from HEK293 cells transfected with the wild-type or mutant minigene L1-GHRexon8-L2. The structure of the correct (L1-Ex8-L2) and aberrant (L1-L2) splice products and their expected sizes are indicated. (B) Schematic representation of the splice events for the wild-type and mutant minigenes. Mutation location is indicated by an asterisk.
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fig3
The IVS7-6 T to A mutation resulted, instead, in the complete skipping of GHR exon 8 and the appearance of the 186nt band, corresponding to exons L1 and L2 joined together, as confirmed by direct sequencing (Fig. 3).