IntroductionThe third enzyme of the heme synthesis pathway, porphobilinogen deaminase (PBGD), is expressed as 2 isoforms: the so-called "housekeeping" form present in all cell types, and a second isoform expressed only in erythroid cells. [1][2][3][4] The autosomal dominant disorder acute intermittent porphyria (AIP) is caused by mutations in the PBGD gene. 5 Clinically, AIP gene variants may or may not affect enzymatic levels within erythroid cells. 3,6,7 One study reported low erythrocyte PBGD activity in blood donors, whereas lymphocyte PBGD activity was within the normal range. 8 A possible mutation within the erythroid-specific part of the PBGD gene was proposed to explain this finding, but was not further explored. On the basis of the extensive genetic studies of AIP, genomic analyses have also shown that PBGD is a pseudogenefree, single-copy gene, thus making it a useful control for RNA analyses. [9][10][11][12][13][14] We sought to use PBGD expression as both an erythroid-specific and housekeeping control for our studies of erythroid-cell gene expression patterns. Unexpectedly, our efforts revealed the existence of 2 distinct erythroid-specific PBGD messenger RNA (mRNA) transcripts.
Study designReverse transcriptase-polymerase chain reaction Total RNA was purified from CD34 ϩ cells collected on specified days after growing in erythropoietin-supplemented media using TRIzol reagent (Life Technologies, Rockville, MD) according to the manufacturer's directions. Reverse transcription (RT) was performed using oligo-(dT) primer and Superscript Reverse Transcriptase (Life Technologies) as suggested by the manufacturer at 42°C for 50 minutes. One microliter of RT reaction was used in 50 L polymerase-chain reaction (PCR) amplification with appropriate primers as follows: initial denaturation at 95°C for 1 minute, denaturing at 95°C for 15 seconds, annealing at 68°C for 15 seconds, and extension at 72°C for 20 seconds for 28 cycles. PCR products were separated in 1.2% agarose gel and stained with ethidium bromide. For PBGD amplification, the following primers were used: 5Ј-ACACAGC-CTACTTTCCAAGCGGAGCCAT-3Ј (primer 1), 5Ј-TGGATCCCTGAG-GAGGGCAGAAG-3Ј (primer 2), 5Ј-TCTTGTCCCCTGTGGTGGACAT-AGCAAT-3Ј (primer 3). Primers 1 and 3 specifically amplify the housekeeping PBGD. Primers 2 and 3 specifically amplify an erythroid PBGD. The PCR products were completely sequenced using a dRhodamine DNA sequencing kit (PE Biosystems, Foster City, CA).
Northern blottingHuman Immune System Multiple Tissue Northern (MTN) Blot II was processed as recommended by the manufacturer (Clontech, Palo Alto, CA). Briefly, the membrane was prehybridized in ULTRAhyb hybridization buffer (Ambion, Austin, TX) at 42°C for 30 minutes. The buffer was replaced with the fresh ULTRAhyb containing 32 P-labeled probe and hybridized at 42°C for 16 hours. The membrane was washed in low-and high-stringency buffers (Ambion) according to the manufacturer's protocol and subjected to autoradiography. Probes specific for the housekeeping (probe H) and an erythroid a...