Britt Thuresson

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.

Genomic typing of the Kidd blood group locus by a single-tube allele-specific primer PCR technique

The Kidd (JK) blood group system is clinically important in transfusion medicine. Alloantibodies to antigens in this system may be produced following blood transfusion or during pregnancy and can result in serious haemolytic transfusion reactions and haemolytic disease of the newborn (HDN).

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.

Wilms’ tumor gene 1 protein represses the expression of the tumor suppressor interferon regulatory factor 8 in human hematopoietic progenitors and in leukemic cells

Wilms’ tumor gene 1 (WT1) is a transcription factor involved in developmental processes. In adult hematopoiesis, only a small portion of early progenitor cells express WT1, whereas most leukemias show persistently high levels, suggesting an oncogenic role. We have previously characterized oncogenic BCR/ABL1 tyrosine kinase signaling pathways for increased WT1 expression. In this study, we show that overexpression of BCR/ABL1 in CD34+ progenitor cells leads to reduced expression of interferon regulatory factor 8 (IRF8), in addition to increased WT1 expression. Interestingly, IRF8 is known as a tumor suppressor in some leukemias and we investigated whether WT1 might repress IRF8 expression. When analyzed in four leukemia mRNA expression data sets, WT1 and IRF8 were anticorrelated. Upon overexpression in CD34+ progenitors, as well as in U937 cells, WT1 strongly downregulated IRF8 expression. All four major WT1 splice variants induced repression, but not the zinc-finger-deleted WT1 mutant, indicating dependence on DNA binding. A reporter construct with the IRF8 promoter was repressed by WT1, dependent on a putative WT1-response element. Binding of WT1 to the IRF8 promoter was demonstrated by chromatin immunoprecipitation. Our results identify IRF8 as a direct target gene for WT1 and provide a possible mechanism for oncogenic effects of WT1 in leukemia.

Clinical utility of immunoglobulin heavy chain gene rearrangement identification for tumour cell detection in multiple myeloma

In an attempt to define the clinical utility of immunoglobulin heavy chain (IgH) gene rearrangement identification for tumour cell detection in multiple myeloma, we investigated 36 consecutive newly diagnosed patients intended for high‐dose chemotherapy in a study protocol. After identification of the IgH rearrangement, an allele specific oligonucleotide (ASO) was constructed and used in a semiquantative PCR for minimal residual disease (MRD) evaluation. The myeloma‐specific IgH gene rearrangement could be identified and an ASO primer constructed in 24 (67%) of the patients. All of these patients underwent transplantation; 22 were autologous, of whom three had PCR‐negative stem cell harvests, and two were allogeneic. 10 patients achieved a clinical complete response (CR) and five were PCR negative in sequential bone marrow analyses. In patients not achieving CR, PCR negativity was occasionally found, but in general the PCR results reflected the clinical status of the patients. No consistent relationship between the bone marrow MRD status and the clinical course was found, and early relapses occurred also in PCR‐negative patients.

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.

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

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.

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 (≤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 ≥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.

A novel B(weak) hybrid allele lacks three enhancer repeats but generates normal ABO transcript levels.

Background and Objectives  Weak expression of A/B histo‐blood group antigens is often explained by single nucleotide substitutions at the ABO locus. However, hybrid alleles containing segments from different ABO alleles can result in unexpected phenotypes and may complicate genotype analysis. We investigated the basis of weak B phenotype in a referred sample.

Granulocyte functions and Neisseria meningitidis: influence of properdin-deficient serum

Granulocyte‐mediated reactions such as opsonization, chemotaxis, and release of granulocyte myeloperoxidase and lactoferrin were studied in properdin‐deficient and normal human serum incubated with serogroup A and W‐135 meningococci. There were no differences between the sera when serogroup A meningococci were studied. Opsonic and chemotactic activity were impaired against serogroup W‐135 meningococci in properdin‐deficient serum. Restitution with properdin restored both activities. We found similar release of myeloperoxidase and lactoferrin from granulocytes challenged with serogroup A or W‐135 meningococci in either sera. These findings are in accordance with the clinical observations of meningococcal infections caused by serogroup W‐135 in properdin‐deficient patients as well as the absence of infections caused by serogroup A meningococci.