Lack of genomic imprinting of DNA primase, polypeptide 2 (PRIM2) in human term placenta and white blood cells

Abstract

PRIM2, encoding a subunit of primase involved in DNA replication and transcription, is expressed in the placenta and is crucial for mammalian development and growth. Its role in placental function is not well understood. Recently, PRIM2 was reported as imprinted in human white blood cells (WBC). We report here our failure to confirm imprinting of the PRIM2 locus in human placenta or WBC. The discordance between our results and those of others are likely due to an incorrectly annotated PRIM2 pseudogene found in the human genome database.

Introduction

Genomic imprinting is an epigenetic process that leads to allelic gene expression in a parent-of-origin specific way.^1-3 Over 100 genes have been conclusively demonstrated to be imprinted in mammals;^4-7 however, both in silico prediction studies and massive parallel sequencing suggest that there may be as many as 1,000 imprinted genes.^8-10 PRIM2, encoding a subunit of primase, is involved in purine and pyrimidine metabolism, and processes such as DNA replication and transcription, crucial for normal growth and development.^11-13 PRIM2 expression has been detected in several tissues including brain, liver, blood, and placenta^14,15 and has been reported as an imprinted, maternally-expressed gene in human white blood cells (WBC).¹⁶ In order to further understand the role of PRIM2 in the placenta we examined whether the PRIM2 gene was imprinted in this organ, as reported for WBC. Here we report lack of PRIM2 imprinting in the human placenta and WBC. PRIM2, presently annotated as NC_000006.11 (NCBI Genome Build 37.1) maps to chromosome 6 whereas a known PRIM2 pseudogene (ref|NW_001838929.1) maps to chromosome 5.^17,18 Our results indicate that the National Center for Biotechnology (NCBI) annotation is incorrect and is likely the cause for incorrectly identifying PRIM2 as imprinted.

Results and Discussion

In an initial sequencing analysis it was noted that the mRNA for Exon 14 of PRIM2 (ref|NM_000947.2) had a higher degree of homology to the region annotated as the PRIM2 pseudogene than to the region annotated as the actual PRIM2. Using this information, primers were developed that could amplify only the PRIM2 pseudogene, or only the PRIM2 transcribed gene (Figs. 1A and 1B). Sequencing of both products confirmed the missannotation in the NCBI database. This was further confirmed by analysis of the whole length of PRIM2 mRNA (data not shown). In both cases the experimentally validated transcribed PRIM2 had a 100% identity to the sequence annotated as the PRIM2 pseudogene. This information allowed us to develop an assay that could examine PRIM2 imprinting by ensuring that only the transcribed PRIM2 gene (tPRIM2 heretofore) was examined.

Figure 1. Ability to discriminate between the PRIM2 pseudogene and the PRIM2 gene. PCR primers were designed to differentially amplify the PRIM2 pseudogene (F3693+R3694) and the transcribed PRIM2 sequence (F3695+R3696). Additional PCR primers were designed to confirm the discrimination. (A). Detection of the PRIM2 pseudogene showing amplification from the placental genomic DNA (gDNA) but not from the placental cDNA indicating that this sequence is not transcribed. B. Detection of the PRIM2 gene showing presence in both the genomic DNA and the cDNA indicating that it is being transcribed. Amplicon identity was confirmed by sequencing as described in text. # refers to sample number.

To examine imprinting, genomic DNA (gDNA) and cDNA were isolated from 21 placentas and tPRIM2 was amplified, sequenced and two single nucleotide polymorphisms (SNPs) identified (rs71214002; A/G and rs62402991; A/G). From these 21 samples, 6 heterozygous individuals were identified. As shown in Figure 2A, heterozygous placentas showed no evidence of imprinting, as both alleles are expressed. Since the only known report of imprinting of PRIM2 in humans was in WBC,¹⁶ WBC were examined and informative tPRIM2 heterozygotes for rs71214002 and rs62402991 were identified and used for further analysis. Results showed that both alleles were expressed at equivalent levels, supporting lack of tPRIM2 imprinting in WBC. Results shown in Figure 2 were confirmed by sequencing at least 12 subcloned PCR products for both gDNA and cDNA. Sequence confirmed expression of both alleles at approximately 50:50 ratio. In an attempt to understand how PRIM2 was reported as imprinted, primers that simultaneously detect both the pseudogene and the transcribed gene were utilized and the amplified DNA analyzed. As shown in Figure 2C this can lead to the incorrect classification of an individual as a heterozygous at the transcribed gene locus, while homozygous at the cDNA level, resulting in evidence of imprinting. What is happening is simultaneous amplification of two targets, the pseudogene and the transcribed gene, that differ in a few nucleotides (thus the “heterozygote” gDNA call). When cDNA is examined, however, only the transcribed “allele” is detected resulting in what appears to be imprinted expression.

Figure 2. Lack of imprinting in the human PRIM2 gene. Individuals with two polymorphisms (rs71214002 and rs62402991) within the coding sequence of the PRIM2 gene were identified and their DNA and cDNA examined to determine whether mono allelic expression indicative of imprinting could be detected. A. Placental samples from informative heterozygotes were collected and DNA and cDNA used to amplify PRIM2 using primers that can differentiate between the transcribed PRIM2 locus and the PRIM2 pseudogene. In all cases examined (n = 6) sequencing results showed that both alleles were transcribed and present in the cDNA when compared with the DNA. This was true for both polymorphisms. B. The same assay but carried on white blood cells (WBC), again showing lack of imprinting of the human PRIM2. C. Sequencing results obtained from amplicons generated by PCR primers that can simultaneously amplify the PRIM2 gene and its pseudogene. The sequencing of the genomic DNA identifies three potential heterozygotes. In the cDNA, however, all heterozygotes disappear suggesting that imprinting is occurring at this locus. This, however, is an artifact caused by amplification of both the PRIM2 gene and its pseudogene, and the presence of polymorphisms between the PRIM2 and the PRIM2 pseudogene.

In summary, while the role of PRIM2 in placental function still needs to be elucidated our results indicate that imprinting disregulation at this locus should not be considered as a plausible explanation for its missexpression in diseased states. This missannotation, in addition to affecting the analysis of imprinting at this locus, affects the mapping of known SNPs to the PRIM2 region due to incorrectly assigning the pseudogene sequence data to the transcribed locus. In other words, some of SNPs in the RefSNP may not be PRIM2 polymorphic alleles among the population or between two parental chromosomes, but between the gene and its pseudogene. This could lead to miscalculations of PRIM2 allelic frequencies in a population and may result in misinterpretation of results.¹⁹

Materials and Methods

Tissue collection

Term placentas were from women who had consented to be part of a study at Duke University Medical Center. Samples were obtained and stored as previously described.²⁰ Whole blood from healthy donors was collected at Durham Regional Hospital. All protocols were approved by the Institutional Review Boards.

Isolation of DNA and RNA, and first-strand cDNA synthesis

Genomic DNA was extracted from placenta and blood using the Wizard Genomic DNA Purification Kit (Promega). RNA from placenta was isolated as previously described.²⁰ For isolation of total RNA from WBC, PAX gene blood RNA system (PreAnalytix) was used and cDNA synthesized from the isolated pure total RNA using Affinity Script™ Multi Temperature cDNA Synthesis Kit (Stratagene).

DNA/cDNA amplification and analysis

Based on the alignment of the pseudogene and the transcribed gene, primers specific to each target were designed. Primers used were F3693 5′-GTGTTGCACTCTGTTGTGTAATTGTGA-3′ and R3694 5′-AGTCTCGTTACCTCAAACCTGCT-3′ for the pseudogene and F3695 5′-GTCACCAAGGCTTAGTGCAGTGA-3′ and R3696 5′-AGTCTCTGTTGTTACCTCAAACCTCCT-3′ for the PRIM2 transcribed sequence. PCR conditions were; during the first 20 cycles, the annealing temperature was decreased each cycle by 0.5°C from 65°C to 55°C, followed by an additional 25 cycles with 55°C annealing temperature. PCR products were subcloned into pCR-Blunt II-TOPO vector (Invitrogen) and sequenced.

Glossary

Abbreviations:

SNP

single nucleotide polymorphism

RefSNP

Reference SNP

WBC

white blood cells