Figure 4. Functional studies of the c.2332–2A>T (p.His778fs) truncating variant.

(A) RT-PCR analysis of PAM exon 21 splicing was performed using blood-extracted RNA from two family members carrying the c.2332–2A>T variant (NIH26 and parent) and four WT control cDNAs. Primers were designed on exons 17 and 22. Both carriers and controls showed a normally spliced transcript (713 bp, upper band), while only the carriers showed an additional alternatively spliced transcript lacking exon 21 (the 613 bp band, identified by the arrow). MW, molecular weight marker. (B) The identity of the PCR products (panel A) was confirmed by Sanger sequencing. The arrows point to the variant-specific peaks present only in the carriers. MUT, mutated; WT, wild-type. (C) Minigene assay. After transfection into HEK-293 AD cells, mRNA synthesis from the plasmids using the cells’ own transcription and splicing machinery led to mRNA products containing (WT) or lacking (variant) exon 21 of PAM flanked by two exons from the pSPL3 vector. The RT-PCR analysis of the minigene transcripts was conducted using vector-specific primers. MW, molecular weight marker; MOCK, cDNA from empty vector-transfected cells consisting of a 260 bp band made up of fragments of pSPL3 exons; --, negative control (RT-PCR without cDNA). The lines separating MOCK and negative control from WT and c.2332–2A>T indicate that data for intervening samples were removed. (D) An expression vector lacking exon 21 of human PAM-1 (H778fs) was transiently expressed in PEAKrapid cells. Proteins were visualized using an antibody to PHMcc (JH246 PHM Ab). Expression of WT PAM produces a major band at 114 ± 1 kDa and a minor one at 105 kDa. The only band visible in the cells expressing p.His778fs migrated at 75 kDa; after signal peptide removal, the mass predicted for this protein – which includes only the first 777 residues of WT PAM-1 but extends 45 residues beyond residue 777 before reaching a stop codon – is 90.31 kDa. NT, not transfected.