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Somatic mosaicism at different stages of ontogenesis
settings, usually regarding the D antigen (and often haplo- typically linked C or E antigens). Apart from inborn forms of chimerism,3 acquired Rh antigen loss was preferentially observed in patients with clonal myeloid diseases,4-11 in some cases with cytogenetic chromosome 1 alterations.12- 14 Also hematologically healthy subjects were observed to have this phenomenon.4,14-18 As the dominant mechanism of acquired Rh phenotype splitting, mosaicism based on myeloid lineage-restricted loss of heterozygosity (LOH) of variable stretches of chromosome 1 was identified with loss of one RH haplotype.4 In one case, somatic RHD mutation was described,19 whereas in other cases, RHD and RHCE gene deletion was reported.4,20,21
In this study, the phenotypic and molecular characteris- tics of spontaneous c antigen anomaly in 2 unrelated indi- viduals were investigated. For the first time, data are pro- vided that demonstrate two different forms of somatic chromosome 1 mosaicism at different stages of ontoge- netic development, as evidenced by involvement of differ- ent cells and tissues. The clinical significance of this phe- nomenon with regard to transfusion medicine and as a potential marker for hemato-oncologic disease is dis- cussed.
Methods
Patients
Two female Caucasoid individuals (proposita A and proposi- ta B, aged 69 and 35 years, respectively) from Switzerland with- out any history of transfusion or hematopoietic stem cell trans- plantation, came to attention with unexplained mixed-field agglutination in routine serological blood group typing. The lat- ter was performed in the course of pretransfusion testing for knee surgery (proposita A) and as part of routine pregnancy monitoring (proposita B). This study was approved by the Swiss Red Cross Institutional Review Board. Written informed consent was obtained for extended testing and inclusion in this investigation.
Serological blood group typing and red cell flow cytometry
Serological blood group typing, anti-erythrocyte antibody screening and direct antiglobulin testing was carried out using gel centrifugation technique (Bio-Rad, Cressier, Switzerland), as described.22 In addition, monoclonal anti-c reagents from Diagast (Loos, France), BAG (Lich, Germany), Immucor (Rödermark) and Ortho Clinical Diagnostics (Neckargemünd, Germany) were used.
Expression of c and C antigens of RBCs from both propositae and of control red blood cell (RBC) samples was determined by flow cytometry (FACSCalibur with CellQuest software, BD Biosciences, San Jose, CA, USA) after indirect immunofluores- cence staining with polyclonal anti-c and anti-C reagents (Molter, Neckargemünd, Germany).
Sorting of nucleated cell subsets from peripheral blood
Cell subsets of ethylenediamine tetraacetic acid (EDTA)-antico- agulated blood samples were quantified and sorted as previously described.4,23
Erythropoietic burst forming unit cultures
Cultures for erythropoietic burst-forming units (BFU-E), scoring and individual clonal picking for subsequent DNA isolation was performed as previously described.4
DNA isolation
Genomic DNA from EDTA-anticoagulated blood was extract- ed with the GenoPrep Cartridge B 350 on a GenoM-6 instru- ment (GenoVision, Vienna, Austria). DNA from buccal swabs, hair samples, finger nails, and single BFU-E colonies with the Qiamp DNA Investigator or Mini Kit (Qiagen, Valencia, CA, USA). DNA from sorted peripheral blood cells was extracted with Chelex.24
Molecular blood group RH genotyping
For RHD and RHCE genotyping, testing for variant RHD alle-
les and RHD zygosity of blood samples, polymerase chain reac- tion (PCR) kits (RBC Ready Gene CDE, Zygofast or RHd, Inno- train, Kronberg, Germany) were used.25 The RHCE*c allele was detected from DNA isolated from single BFU-E colonies with sequence specific monoplex real-time PCR using primers, probes and real-time PCR reagents as previously described,26 with a modified cycle protocol for increased sensitivity.
Microsatellite analysis
DNA prepared from whole blood and hair roots was tested in a multiplex-PCR of 15 highly polymorphic autosomal microsatellite loci to check for the existence of a possible chimerism (AmpFlSTR IDentifiler PCR Amplification Kit, Applied Biosystems, Foster City, CA, USA).
DNA samples from whole blood, buccal swabs (only proposi- ta B), single hairs roots, nucleated blood cell subsets, or BFU-E colonies were analyzed with up to 16 different primer pairs tar- geting polymorphic dinucleotide microsatellite markers located on chromosome 1.
Fluorescence in situ hybridization analyses
Dual-color fluorescence in situ hybridization (FISH) analyses on fixed peripheral blood cells of both propositae were per- formed as previously described.4 P1-based artificial chromosome clones that encompass the RHD/RHCE and AF1q gene loci, respectively, were used. At least 200 cells per proband were scored and the signal patterns recorded separately for segmented
and round nuclei.
Results
Spontaneous Rh blood group anomaly in 2 unrelated individuals
Routine serological blood group determination revealed unexpected mixed-field agglutination with respect to c antigen typing in 2 unrelated females without known hematologic disorder (proposita A and B). This was evi- dent with all employed anti-c typing reagents (six mono- clonal and one polyclonal). The proportion of c-positive red cells by flow cytometry was 53% and 50% in proposita A and B, respectively (Table 1). Apart from this, both individuals showed a normal C+D+E-e+ Rh pheno- type. All other tested blood groups (ABO, MNS, P1Pk, Lutheran, Kell, Duffy, Kidd) were of normal phenotype (Table 1). No unexpected red cell antibodies were found in the plasma of these individuals, and the direct antiglob- ulin test with their erythrocytes was negative.
Routine RHD/RHCE genotyping combined with RHD zygosity determination of blood-derived DNA from both propositae yielded RHD heterozygosity (Dd) and predict- ed common Ccee phenotypes.
The c antigen quantities of their c-positive RBC subsets were similar to CcDdee phenotype control RBCs (Figure
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