From www.bloodjournal.org by guest on December 29, 2014. For personal use only. CORRESPONDENCE Decreased Stability of the 0 Allele mRNA Transcript of the AB0 Gene To the Editor: pectedly, the weexpression markedly reduced of observed 0 allele in normal A 0 or BO genotype individuals. Mononuclear cells from seven adult bone marrows and15 fetal We previously reported a method for genotyping the major alleles umbilical cord bloods were examined for A B 0 expression. RNA of the A B 0 bloodgrouplocus.’ We subsequentlydeveloped a rewasisolated,reverse-transcribedwithrandomhexamersandamversetranscription-polymerasechainreaction (RT-PCR) methodtoplifiedby PCR. The primers used amplify nucleotides 12 through 329 of the A B 0 cDNA sequence’,’ spanning the site at position assess the relative expression of the alleles of the A B 0 gene. Unex- Blood, Vol 87, No 7 (April I), 1996:3061 pp 3061-3068 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. CORRESPONDENCE 3062 261 where the nonfunctional 0 allele has a single base deletion.' The deletion creates a Kpnl restriction endonuclease site in the 0 allele, where there is a BstEIl site present in the A and B alleles. Ten microliter aliquots of the RT-PCR product were digested with 10 units of Kpnl or BstEII and resolved on 8 8 polyacrylamide gels (Fig I ) . Digestion of the 316bp 0 allele RT-PCR product with KpnI results in a band of 249bp. whereas no digestion of the product occurs with BstEII. Likewise, digestion of the A or B alleles with l 2 3 4 5 6 7 8 9 1 0 BsrEIl results in a 249bp product, and there is no digestion with Kpnl. The five 00 genotype individuals all expressed the 0 allele Fig 1. A B 0 RT-PCR product after diagnostic restriction enzyme and the two A A genotype individuals both expressed the A allele. digestion. cDNA from five individuals whose genotypes were 00 However, when the ten A 0 and five BO genotype individuals were (lanes l,2). A 0 (lanes 3, 4). A 0 (lanes 5, 6). BO (lanes 7, 81 and AA examined, the amount of the 0 allele was considerably diminished (lanes 9,101 was amplified. RT-PCR products were digested with Kpnl (lanes 1.3, 5, 7 , 91, or Esdll (lanes 2, 4, 6, 8, 10). relative to that of the A or B allele (see lanes 3 through 8, Fig I ) . Thus, although the 0 allele is expressed in 00 genotype individuals, the majority of the steady-state mRNA of the AB0 gene is comprised of the A or B allele in individuals heterozygous of thetranscriptionalregulation of the AB0 geneisrequiredto for the 0 allele. Similar patterns of expression were also seen for understand the mechanisms underlying these observations. the hematopoietic and colon carcinoma cell lines; KC122, LIM2412 (genotype AO), LIM 1215 (BO), and K562, HEL. and LIM 2405 (00). ACKNOWLEDGMENT RT-PCR products from A 0 and BO heterozygotes digested We thank Annette Hogg fordiscussionandR.E. Sage for his with BsrEIl gave a residual undigested fraction whereas AA RTinvaluable support. PCR products digested completely. This result was repeatable, and consistent with the presence of a small amount of 0 allele. This result and the apparent absence of a digested band correDenise S . O'Keefe sponding to the 0 allele in some KpnI digests could be explained Alexander Dobrovic by the formation of heteroduplexes. The small amount ofPCR Department of H a e r n a t n l o ~ ~ ~ / O n ~ ~ ~ l ~ ~ product corresponding to the 0 transcript would almost entirely The Queen Elizuheth Hospiral be found in heteroduplexes. These heteroduplexes would be resisDepartment of Medicine tant to digestion with KpnI and would remain in the undigested Universie of Adeluide band corresponding to A or B. Woodville. Austrnlia The markedly reduced steady-state 0 allele mRNA level in heterozygotesmaybeexplainedbylowerstabilityofthe 0 transcript. The 0 allele has a single base deletion that leads to a premature stop codon at REFERENCES nucleotide 352, and subsequent early termination of translation." Apparent I . O'Keefe DS, Dobrovic A: A rapid and reliable method for instabilityofmRNAtranscriptswithprematurestopcodonshas been genotyping the AB0 blood group. Hum Mutat 2:67, 1993 documentedfor a numberofgenes in a widerangeofspecies. The 2. Yamamoto F, Marken J. Tsuji T, White T, Clausen H, Hakomechanisms behind this phenomenon are not completely ~nderstood.~.~.~ mori S: Cloningandcharacterization ofDNA complementary to The decreased levels are not due to differential transcription as mutant humanUDP-GalNAc:Fuca I 2Galal -t 3GalNActransferase and normal alleles are transcribed at the same rate, and the loss of the (histo-blood group A transferase) mRNA. J Biol Chem 265:1146, transcript with the premature stop codon has been shown to take place I990 after ~plicing.'"~ Interestingly, reduced levels of the 0 allele were also 3. Yamamoto F Review: Recent progress in the molecular geseen by us in a C O 2 heterozygote (data not shown). The transcript of netic studyof the histo-blood groupAB0 system. Immunohaematolthe O2allele has no premature stop codon' and thus would be expected ogy 1 0 1 . 1994 to have similar stability to the A and B alleles. 4.Yamamoto F, Clausen H, WhiteT,Marken J, Hakomori S: The results seen hereforthe AB0 genestrikinglyresemblethose Molecular genetic basis of the histo-blood group AB0 system. Nareported for the retinoblastoma tumour suppressor gene (RB l).5 Whereas ture 345:229, 1990 thenormalRBIallelewasexpressed in thelymphocytesofpatients 5. Dunn JM. Phillips RA, Becker A, Zhu X, Gallie BL: Mutations carrying germline mutations resulting in premature stop codons, the exin the RBI geneandtheir effects on transcription. Mol Cell Biol in tumors pressionofmutantRBIalleleswasnotdetected.However, 9:4956,1989 where the normal allele wa. physically lost, the mutant allele waseasily 6. Cheng J, Maquat LE: Nonsense codons can reduce the abundetectable. The authors proposed that transcription of the mutant allele dance of nuclear mRNA without affecting the abundance of prewas i n c r e a d to compensate for loss of the functional product. mRNA or thehalf-life of cytoplasmic mRNA.Mol Cell Biol Similarly, it seems that the stability of the 0 mRNA is reduced, 13:I 892, I993 and therefore it could be proposed that the amount of transcript, or 7. Belgrader P, Cheng J, Zhou X, Stephenson LS, Maquat LE: functional gene product, either directly or indirectly regulates AB0 Mammalian nonsense codons canbe cis effectors of nuclear mRNA transcription. In 00 genotype individuals, transcription of the gene half life. Mol Cell Biol 14:8219, 1994 would be increased to compensate for the decreased stability of the 8. Yamamoto F, McNeill PD. Yamamoto M, Hakomori S, 0 mRNA. This is consistent with Northern analysis which shows Bromilow IM, Duguid JKM: Molecular genetic analysis of the AB0 blood group system: 4. Another type of 0-allele. Vox Sang approximately equivalent amounts of AB0 mRNA in cell lines de64: 175, I993 rived from A,B,and 0 blood group individuals.' Further analysis + From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 1996 87: 3061-3062 Decreased stability of the O allele mRNA transcript of the ABO gene [letter] DS O'Keefe and A Dobrovic Updated information and services can be found at: http://www.bloodjournal.org/content/87/7/3061.2.citation.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.