Large deletions involving the regulatory upstream regions of A4GALT give rise to principally novel P1PK-null alleles.

BACKGROUND: Cells of the clinically important p histo-blood group phenotype lack P1, P(k) , and P glycosphingolipid antigens. All cases investigated so far are due to alterations in the 4-α-galactosyltransferase-encoding Exon 3 of A4GALT. Repetitive elements in the genome can mediate DNA rearrangements, the most abundant being the Alu family of repeats. STUDY DESIGN AND METHODS: The aim of this study was to determine the genetic basis of three p samples with intact A4GALT open reading frames, using long-range polymerase chain reaction (PCR) and sequencing. In addition, transcript measurements were performed with quantitative PCR. RESULTS: This is the first report of the p phenotype as the result of large deletions in A4GALT, comprising the proposed promoter and noncoding Exons 1 and 2a. The breakpoints were different in all three samples and revealed the presence of Alu or MIRb sequences directly flanking, or in close proximity to, all junctions. Furthermore, no A4GALT transcripts could be detected. CONCLUSION: In summary, our data elucidate a new explanation underlying the p phenotype, implicating the deleted regions of A4GALT as crucial for P1 and P(k) synthesis, possibly due to loss of binding sites for erythroid transcription factors. Furthermore, analysis of these regions will improve genetic blood group prediction.

Identification of a novel A4GALT exon reveals the genetic basis of the P1/P2 histo-blood groups.

The A4GALT locus encodes a glycosyltransferase that synthesizes the terminal Galα1-4Gal of the P(k) (Gb3/CD77) glycosphingolipid, important in transfusion medicine, obstetrics, and pathogen susceptibility. Critical nucleotide changes in A4GALT not only abolish P(k) formation but also another Galα1-4Gal-defined antigen, P1, which belongs to the only blood group system for which the responsible locus remains undefined. Since known A4GALT polymorphisms do not explain the P1-P(k)+ phenotype, P(2), we set out to elucidate the genetic basis of P(1)/P(2). Despite marked differences (P(1) > P(2)) in A4GALT transcript levels in blood, luciferase experiments showed no difference between P(1)/P(2)-related promoter sequences. Investigation of A4GALT mRNA in cultured human bone marrow cells revealed novel transcripts containing only the noncoding exon 1 and a sequence (here termed exon 2a) from intron 1. These 5'-capped transcripts include poly-A tails and 3 polymorphic sites, one of which was P(1)/P(2)-specific among > 200 donors and opens a short reading frame in P(2) alleles. We exploited these data to devise the first genotyping assays to predict P1 status. P(1)/P(2) genotypes correlated with both transcript levels and P1/P(k) expression on red cells. Thus, P(1) zygosity partially explains the well-known interindividual variation in P1 strength. Future investigations need to focus on regulatory mechanisms underlying P1 synthesis.

P1/P2 genotyping of known and novel null alleles in the P1PK and GLOB histo-blood group systems.

BACKGROUND: The rare but clinically important null phenotypes of the P1PK and GLOB blood group systems are due to alterations in A4GALT and B3GALNT1, respectively. A recently identified single-nucleotide polymorphism in Exon 2a of A4GALT predicts the common P1 and P2 phenotypes but rare variants have not been tested. STUDY DESIGN AND METHODS: The aim of this study was to analyze 84 p, P1 (k) , and P2 (k) samples, with special emphasis on unknown alleles and the P(1) /P(2) marker. Of these, 27 samples came from individuals not previously investigated genetically and were therefore subjected to sequencing of A4GALT or B3GALNT1, and a subset was tested by flow cytometry. RESULTS: The P(1) /P(2) genotyping linked 20 p-inducing mutations in A4GALT to P(1) or P(2) allelic background. Eight p alleles remain unlinked due to compound heterozygosity. For 23 of 25 P(k) samples, concordant results were observed: P1 (k) samples had at least one P(1) allele while P2 (k) had P(2) only. The two remaining samples typed as P1+ and P1+(w) but were genetically P(2) /P(2) . A tendency toward higher P(k) antigen expression was observed on P1 (k) cells compared to P2 (k) . In total, six previously unknown null mutations were found and characterized in A4GALT while four new changes were revealed in B3GALNT1. CONCLUSION: For the first time, p alleles were shown to occur on both P(1) and P(2) allelic backgrounds. Furthermore, P(1) /P(2) genotyping predicted the P1 (k) versus P2 (k) phenotype in more than 90% of globoside-deficient samples. The number of GLOB-null alleles was increased by 50% and several P1PK-null alleles were identified.

Minimal residual disease assessment in childhood acute lymphoblastic leukaemia: a Swedish multi-centre study comparing real-time polymerase chain reaction and multicolour flow cytometry.

Minimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi-centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow-up samples in 228 children using real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0·1%, which was the sensitivity level reached in all analyses, the concordance between RQ-PCR and FCM MRD values at day 29 was 84%. In B-cell precursor ALL, an MRD level of ≥0·1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ-PCR, a higher MRD cut-off (≥0·2%) improved the predictive capacity of RQ-PCR. In T-ALL, RQ-PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ≥0·1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ-PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.

Applicability of IG/TCR gene rearrangements as targets for minimal residual disease assessment in a population-based cohort of Swedish childhood acute lymphoblastic leukaemia diagnosed 2002-2006.

Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukaemia (ALL) treatment protocols. Here, we aimed to address the applicability of rearranged antigen-receptor genes as potential MRD markers using real-time quantitative polymerase chain reaction (RQ-PCR) in a Swedish population-based cohort. From 334 childhood ALL cases diagnosed during 2002-2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes. Allele-specific oligonucleotides were designed, and the sensitivity and quantitative level was determined for each target. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B-cell precursor ALL (BCP ALL) and 94% (33/35) of T-ALL. A sensitive RQ-PCR analysis (< or = 10(-4)) was obtained in 89% (216/244) of BCP ALL and in 74% (26/35) of T-ALL, whereas two sensitive targets were only available in 47% (115/244) of BCP ALL and 29% (10/35) of T-ALL cases. With the stratification threshold of > or = 10(-3), which is applied in the current Nordic treatment protocol (NOPHO-ALL 2008) for the identification of high-risk patients, 93% of BCP ALL and 86% of T-ALL reached this quantitative range by at least one target gene. Taken together, this national retrospective study demonstrates that an IG/TCR target for MRD monitoring can be identified in the majority of childhood ALL cases, whereas identification of a second sensitive target gene needs to be improved.

Homozygosity for a null allele of SMIM1 defines the Vel-negative blood group phenotype.

The Vel antigen is present on red blood cells (RBCs) from all humans except rare Vel-negative individuals who can form antibodies to Vel in response to transfusion or pregnancy. These antibodies may cause severe hemolytic reactions in blood recipients. We combined SNP profiling and transcriptional network modeling to link the Vel-negative phenotype to SMIM1, located in a 97-kb haplotype block on chromosome 1p36. This gene encodes a previously undiscovered, evolutionarily conserved transmembrane protein expressed on RBCs. Notably, 35 of 35 Vel-negative individuals were homozygous for a frameshift deletion of 17 bp in exon 3. Functional studies using antibodies raised against SMIM1 peptides confirmed a null phenotype in RBC membranes, and SMIM1 overexpression induced Vel expression. Genotype screening estimated that ~1 of 17 Swedish blood donors is a heterozygous deletion carrier and ~1 of 1,200 is a homozygous deletion knockout and enabled identification of Vel-negative donors. Our results establish SMIM1 as a new erythroid gene and Vel as a new blood group system.

ABO transcript levels in peripheral blood and erythropoietic culture show different allele-related patterns independent of the CBF/NF-Y enhancer motif and multiple novel allele-specific variations in the 5'- and 3'-noncoding regions.

BACKGROUND: Mechanisms regulating the ABO gene are unclear, especially in the hematopoietic compartment. The number of 43-bp repeats in the CBF/NF-Y-binding enhancer region is considered to have a major influence on transcription. STUDY DESIGN AND METHODS: Transcript levels in peripheral blood and in erythropoietic culture of CD34+ cells from marrow donors were measured with TaqMan assays. The 5'-regulatory region and 3'-downstream sequences were investigated to determine if allelic variations occur. RESULTS: Surprisingly, transcripts from A(1) and A(2) alleles could not be detected in peripheral blood, although transcripts from B/O(1)/O(1v)/O(2) alleles were readily observed. Sequencing of approximately 4 kb upstream and 1.8 kb downstream of the coding region showed multiple novel allele-specific and allele-related motifs. No correlation between these sequence variations and transcript levels was found, however. Contradictory to the results with peripheral blood, in erythropoietic culture of CD34+ cells from healthy marrow donors transcripts from A(1) and A(2) alleles were found at higher levels than transcripts from B/O(1)/O(1v) alleles. CONCLUSION: These data do not support previous suggestions that nonsense-mutated O(1)/O(1v) transcripts are eliminated first. Furthermore, our results contradict the notion that the number of repeats in the upstream CBF/NF-Y-binding enhancer region, which contains four 43-bp repeats in A(2)/B/O(1)/O(1v) but only one 43-bp unit in A(1)/O(2) alleles, determines the transcription rate. The reason for the remarkable discrepancy between blood and marrow remains to be elucidated.