Figure 3

Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. A. Agarose gel of RT-PCR reaction products from LINS cDNA amplification in a control (con), parents (I1, I2) and patients (II1, II2) compared to a DNA 100bp ladder (M). The gel showed a ~1014bp band of the wild type LINS transcript encompassing exon 5 in a normal control (con) accompanied with multiple isoforms of varying length (~1000bp). In patients (II1, II2) lanes, only smaller bands were seen (~400bp) suggesting a homozygous deletion of around 600bp. The parents (-I1, -I2) have both upper and lower bands suggesting that they carry the 600bp deletion in a heterozygous state. B. A schematic diagram of the splicing defect seen in patients based on Sanger sequencing data of the cDNA. The upper most band of the higher and lower bands seen in RT-PCR gel were purified and sequenced. This higher band noticed in control and parents was found to include exons 3,4,5 and 6. On the other hand, Exon 5 (E5) was found to be missing in the lower-size band seen in both patients and parents. These results suggested that the genomic deletion at the end of E5 abolished a canonical splicing site masking the exon from the splicing machinery which considered it to be part of intron 4 and cut it out of the nascent mRNA. C. Analyzing the accompanying upper and lower bands amplified by RT-PCR suggested the presence of at least 3 LINS transcripts alternatively spliced in exon 6 with the putative multiple splice junctions are shown. All the lower bands lack exon 5 while the upper bands include it compared to the RefSeq NM_001040616.2.