Human erythroid porphobilinogen deaminase exists in 2 splice variants

Abstract

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...

Multiplex mRNA profiling for the identification of body fluids

“…PCR Primer sequences for STATH and HTN3 were designed using Oligo 1 Primer Analysis Software, Version 6 (Lifescience Software Resource, Long Lake, MN). PCR Primer sequences for PBGD, PRM1, and PRM2 were obtained from published sources [12,13]. Table 1 shows the PCR primer sequences and the expected product sizes.…”

“…parallel) analysis procedures for body fluid identification that are compatible with current DNA analysis procedures, we initially considered assays based upon proteins and messenger RNA (mRNA) since both are expressed in a tissue-specific manner. However, multiplex analysis of complex, partially www.elsevier.com/locate/forsciint Forensic Science International 152 (2005) [1][2][3][4][5][6][7][8][9][10][11][12] degraded protein mixtures, such as those present in body fluid stains, awaits further developments in proteomics. Messenger RNA is considered a better option at present because the technologies for massively parallel analysis continue to be developed due to rapid advances in the field of functional genomics.…”

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

“…Blood was chosen for its easy availability, stability and the number of reported markers [6][7][8][9][10][13][14][16][17][19][20]. The following blood markers were included in the study: HBA and HBB are the alpha-and betasubunits of hemoglobin A [21,22]; aminolevulinate synthase 2 (ALAS2) is an erythroid-specific mitochondrial enzyme, that catalyzes the first step in the heme biosynthetic pathway [23]; CD3 gamma molecule (CD3G) is part of the T-cell receptor-CD3 complex, which plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways [24]; ankyrin 1 (ANK1) is an erythrocyte membrane protein, which provides the primary linkage between the membrane skeleton and the plasma membrane [25]; the erythroid form of porphobilinogen deaminase (PBGD) is the third enzyme of the heme biosynthetic pathway [26]; β-spectrin (SPTB) is an erythrocyte membrane protein [27]; Aquaporin (AQP9) is a water channel protein expressed in peripheral leukocytes [28]. AQP9 was chosen as one representative out of nine, all of which were stated by the authors [13] to be co-expressed in vaginal secretion.…”

Nine mutations including three novel mutations among Russian patients with acute intermittent porphyria

“…Erythroid mRNA mainly includes exons 2-15, and non-erythroid mRNA exons 1 and 3-15 (Grandchamp et al, 1987;Gubin and Miller, 2002).…”