Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype.

BACKGROUND: We previously showed that several variations in the RHD gene, including synonymous changes, can be classified as splice site variants and may play a direct role in D variant phenotype expression. We sought to extend our study to additional candidates, notably in the first and last exons of the gene, by engineering a novel universal splice reporting vector, i.e., minigene. STUDY DESIGN AND METHODS: Our previous plasmid construct was modified to allow subcloning of any exon(s) of interest for assessing effect of variations on splicing. Seventeen novel and/or uncharacterized variations of the RHD gene were selected for the study and tested in our novel model. RESULTS: We engineered and validated a novel universal minigene for assessing virtually any variations of interest for splicing defect. Of the 17 variants tested in the novel model, 11 were shown to alter splicing either totally or partially, including the silent c.1065C>T variation, which induces major skipping of exon 7, and may therefore be responsible for reducing D antigen expression. We also showed that while all three missense variations c.1154G>C, c.1154G>T, and c.1154G>A in exon 9 are splice site variants, splicing is differentially altered and D-negative phenotype observed in the presence of the latter substitution is likely due to a defect in RhD protein folding. CONCLUSION: Overall, we hypothesize that splicing alteration is likely to be a common mechanism of D phenotype variation that has been underestimated so far. Further large-scale studies are necessary to demonstrate this statement definitely.

A RHAG point mutation selectively disrupts Rh antigen expression.

OBJECTIVES: The aim of this study was to characterise a novel mutation in the gene encoding RhAG in order to elucidate a molecular mechanism for Rh antigen expression and spherocytosis. BACKGROUND: Rhesus-associated glycoprotein (RhAG) is critical for maintaining the structure and stability of erythrocytes. Single missense mutations in the gene encoding RhAG are sufficient to induce spherocytosis and deficiencies in Rh complex formation. We report a novel missense mutation that incompletely disrupts Rh antigen expression and selectively knocks out RhD antigen expression. METHODS: Blood samples were taken from a 38-year-old male, his brother, his wife and his daughter in Xi'an, China. To detect the proband's RhAG and D antigen expression, the RBC were stained with anti-D and anti-RhAG and analysed by flow cytometry. Red blood cell morphology was detected with atomic force microscopy (AFM). Genomic DNA was isolated from whole blood samples, and the RHD, RHCE and RHAG alleles were sequenced and analysed. The mutation was mapped onto a predicted crystal structure of RhAG by the I-TASSER server and visualised using PyMOL. RESULTS: Morphological testing by AFM found clear evidence of spherocytosis in the proband's erythrocytes. RHAG gene sequencing identified the mutation at sequence 236G > A, resulting in a serine to asparagine substitution at residue 79 (S79N). Family survey indicated that inheriting this allele is necessary and sufficient to cause the condition. Mapping the mutation onto a predicted crystal structure of RhAG revealed the proximity of the mutation to the critical structural elements of the protein. CONCLUSIONS: A novel RHAG mutation significantly lowers RhAG antigen expression and antigen-mediated agglutination intensity.

Two large deletions extending beyond either end of the RHD gene and their red cell phenotypes.

Only two partial deletions longer than 655 nucleotides had been reported for the RHD gene, constrained within the gene and causing DEL phenotypes. Using a combination of quantitative PCR and long-range PCR, we examined three distinct deletions affecting parts of the RHD gene in three blood donors. Their RHD nucleotide sequences and exact boundaries of the breakpoint regions were determined. DEL phenotypes were caused by a novel 18.4 kb deletion and a previously published 5.4 kb deletion of the RHD gene; a D-negative phenotype was caused by a novel 7.6 kb deletion. Examination of the deletion-flanking regions suggested microhomology-mediated end-joining, replication slippage, and non-homologous end-joining, respectively, as the most likely mechanisms for the three distinct deletions. We described two new deletions affecting parts of the RHD gene, much longer than any previously reported partial deletion: one was the first deletion observed at the 5' end of the RHD gene extending into the intergenic region, and the other the second deletion observed at its 3' end. Large deletions present at either end are a mechanism for a much reduced RhD protein expression or its complete loss. Exact molecular characterization of such deletions is instrumental for accurate RHD genotyping.

Rhesus CE expression on patient red blood cells is an independent prognostic factor for adenocarcinoma of the lung.

OBJECTIVES: The influence of blood group antigens on cancerogenesis is shown for distinct tumor types, yet the impact of Rhesus blood group antigens in lung cancer is not clarified. MATERIALS AND METHODS: To investigate the impact of Rhesus blood groups a non-small cell lung cancer (NSCLC) collective (n = 1047) was analyzed retrospectively. Using a second cohort of n = 340 primarily operated stage I-III NSCLC patients, we evaluated immunohistochemistry of CD47-antibody stained tissue samples in correlation to histopathologic subtype and Rhesus blood group. RESULTS AND CONCLUSION: In 516 of 1047 patients blood group data were available. Seven different RhCE phenotypes were grouped as "··ee," "ccE·," and "C·E·." Adenocarcinoma patients with Rh "··ee" revealed improved overall survival (29 (21.2-36.8) m; HR 1.00 [index]) compared with Rh "ccE·" (19 (1.9-36.1) m; HR 1.76 [1.15-2.70]) and Rh "C·E·" (10 (7.4-12.6) m; HR 2.65 [1.70-4.12]) univariately (P < .001) and multivariately (P < .001). Rh "··ee" showed reduced incidence of CNS-metastasis (P = .014) and metastasis count (P = .032) in stage IV adenocarcinoma. Immunohistochemistry associated CD47-positivity with adenocarcinomas (n = 340, P = .048). In n = 51 cases blood group data were available. The prognostic effect of Rh "··ee" compared with Rh "ccE·" and Rh "C·E·" was stated (P = .001), foremost in CD47-positive adenocarcinomas (Rh "··ee" vs. Rh "ccE·" and Rh "C·E·," P = .008). Inversely Rh "ccE·" or Rh "C·E·" was found beneficial in CD47-negative non-adenocarcinomas (P = .046). Phenotypic RhCE expression may be an independent prognostic factor for overall survival in adeno-NSCLC. We hypothesize an erythrocytic-immunologic interaction with tumor tissue, possibly altered by RhCE and CD47, resulting in a metastatic prone condition.

From genetic variability to phenotypic expression of blood group systems.

More than 300 red blood cell (RBC) antigens belonging to 36 blood group systems have been officially reported in humans by the International Society of Blood Transfusion (ISBT). Phenotypic variability is directly linked to the expression of the 41 blood group genes. The Rh blood group system, which is composed of 54 antigens, is the most complex and polymorphic system. Many rare genetic variants within the RH (RHD and RHCE) genes, involving various mutational mechanisms (single-nucleotide substitutions, short insertions/deletions, rearrangements, large deletions), have been reported in the literature and reference databases. Expression of the variants induces variable clinical outcomes depending on their nature and impact on antigen structure. Their respective molecular and cellular effects remain however poorly studied. Biological resources to conduct this research are also barely available. We have paid a specific attention to three different classes of single-nucleotide substitutions: 1/ splice site variants in the Rh, Kell, Kidd, Junior and Langereis systems by the minigene splicing assay developed locally; 2/ missense variants in the RhD protein and their effect on intermolecular interaction with its protein partner RhAG, intracellular trafficking and plasma membrane integration; and 3/ synonymous variants in the RHD gene. Overall not only this project has fundamental objectives by analyzing the functional effect of variants in order to make genotype-phenotype correlation, but the aim is also to develop/engineer molecular tools and cell models to carry out those studies.

RHD zygosity predicts degree of platelet response to anti-D immune globulin treatment in children with immune thrombocytopenia.

Anti-D immunoglobulin is a common front-line treatment for childhood immune thrombocytopenia (ITP) that typically results in a rapid and significant increase in platelet count. Unpredictable treatment responses and interpatient variability limit more widespread use. We hypothesized that anti-D response variability is influenced by RHD gene zygosity and erythrocyte D antigen expression. We compared RHD zygosity and quantitative D antigen expression to anti-D treatment results. Hemizygous RHD subjects demonstrated significantly higher platelet increases and peak platelet counts than homozygous RHD subjects. Future studies should investigate the mechanisms by which RHD zygosity and D antigen expression affect platelet responses to anti-D immunoglobulin.

Identification of FoxM1/Bub1b signaling pathway as a required component for growth and survival of rhabdomyosarcoma.

We identified Bub1b as an essential element for the growth and survival of rhabdomyosarcoma (RMS) cells using a bar-coded, tetracycline-inducible short hairpin RNA (shRNA) library screen. Knockdown of Bub1b resulted in suppression of tumor growth in vivo, including the regression of established tumors. The mechanism by which this occurs is via postmitotic endoreduplication checkpoint and mitotic catastrophe. Furthermore, using a chromatin immunoprecipitation assay, we found that Bub1b is a direct transcriptional target of Forkhead Box M1 (FoxM1). Suppression of FoxM1 either by shRNA or the inhibitor siomycin A resulted in reduction of Bub1b expression and inhibition of cell growth and survival. These results show the important role of the Bub1b/FoxM1 pathway in RMS and provide potential therapeutic targets.

Molecular background of D-negative phenotype in the Tunisian population.

BACKGROUND: Most studies of the molecular basis of Rhesus D-negative phenotype have been conducted in Caucasian and African populations. A comprehensive survey of RHD alleles was lacking in people from North Africa (Tunisians, Moroccans and Algerians) which could be very efficient for managing donors and patients carrying an RHD molecular variant. We analyse the molecular background of D-negative population in Tunisia in the present study. MATERIALS AND METHODS: Blood samples were collected from native Tunisians. A total of 448 D-negative donors from different regions of Tunisia were analysed by RHD genotyping according to an adopted strategy using real-time PCR, ASP-PCR and sequencing. RESULTS: Among the 448 D-negative samples, 443 were phenotyped unequivocally as true D-negative including three molecular backgrounds which were RHD gene deletion (n = 437), RHDψ pseudogene (n = 2) and RHD-CE-D hybrid gene (n = 4) with the respective frequencies of 0·9900, 0·0023 and 0·0046. The remaining five samples, in discordance with the serological results, were identified as two weak D type 11, one weak D type 29, one weak D type 4·0 and one DBT-1 partial D. CONCLUSION: This study showed that the Tunisian population gets closer to Caucasians, given that the RHD gene deletion is the most prevalent cause of D-negative phenotype, but it is slightly different by the presence of the RHDψ pseudogene which was found with a very low frequency compared with that described in the African population. Nevertheless, the relative occurrence of weak D variants among studied serologically D-negative samples make necessary the adaptation of RHD genotyping strategy to the spectrum of prevalent alleles.

On the trail of anti-CDE to unexpected highlights of the RHD*weak 4.3 allele in the Upper Austrian population.

BACKGROUND AND OBJECTIVES: The application of a commercial available microcolumn system for ABO/RH determination lead to irregular results in CDE typing of seemingly D- blood samples. In this study, we introduce a comprehensive serological and molecular work-up of a novel haplotype carrying the RHD*weak 4.3 in combination with an aberrant RHCE*ce. MATERIALS AND METHODS: The molecular background was characterized by RHD and RHCE-specific DNA sequencing, RHD cDNA sequencing and RHD zygosity testing. Haplotype-specific extraction and inheritance analysis were initiated to determine the linkage of the polymorphisms. The genetic admixture was studied by whole genome SNP array analysis. Serology was done using commercial available standard techniques and by in-house sera likewise. RESULTS: All samples (n = 29) were shown to harbour an altered RHD(T201R, F223V, P291R) allele known as RHD*weak 4.3 associated with a RHCE*ce(W16C, A36T, L245V) gene formation, expressing C(X) and VS. Both anti-C(X) and anti-V/VS were detected as contaminating antibodies in a commercial available microcolumn system for ABO/RH determination accounting for the positive results in CDE typing. Compared with other population data, the samples were clearly identified as Caucasian. CONCLUSION: The RHD*weak 4.3 allele with markedly reduced antigen D expression was shown to be associated with an altered RHCE gene formation leading to the expression of C(X) and VS. Its frequency was estimated 1 in 854 among apparently D- Upper Austrian blood donors.

Expression of the Rh/RhAG complex is reduced in Mi.III erythrocytes.

BACKGROUND AND OBJECTIVES: Miltenberger blood group antigen subtype III (Mi.III) is characterized by expression of a glycophorin B-A-B hybrid (Gp.Mur) on the erythrocyte surface. The two alleles of glycophorin B are substituted with the B-A-B hybrid alleles in homozygous Mi.III (Mi.III(+/+)), and thus, Mi.III(+/+) erythrocytes lack glycophorin B (GPB) and express Gp.Mur only. Because GPB is a major component of the Rh complex on RBCs, in this study, we explored how the absence of GPB might affect Rh expression in Mi.III RBCs. MATERIALS AND METHODS: (1) Mi.III+ RBCs were serologically identified and further differentiated their homozygosity or heterozygosity by immunoblot or direct sequencing. (2) RhD and RhCcEe mRNA was cloned, and their sequences analysed. (3) The expression levels of Rh antigen, Rh-associated glycoprotein (RhAG) and the U antigen in MI.III vs. non-Mi.III RBCs were assessed by flow cytometry and Western blot. RESULTS: Compared with the non-Mi.III samples, the surface expression of the Rh antigen was reduced to 76·4% in Mi.III(+/+) RBCs and 93·6% in Mi.III(+/-). RhAG expression was also significantly reduced in Mi.III(+/+), but not in Mi.III(+/-). The U antigen expression in Mi.III(+/-) was only 14·9% relative to the control RBCs, while GPB was half the level of the controls. The mRNA sequences of Rh polypeptides from Mi.III+ samples were identical to the NCBI reference sequences. CONCLUSION: Substitution of GPB with Gp.Mur significantly reduced the expression of Rh antigen and RhAG on the Mi.III(+/+) erythrocyte membrane. The Mi.III phenotype is predicted to induce considerable structural variations within the band 3/Rh-associated macrocomplexes.

RHCE*ceAR encodes a partial c (RH4) antigen.

Thr Rh blood group system is highly complex both in the number of discreet antigens and in the existence of partial antigens, especially D and e. Recently, several partial c antigens have been reported. Here we report findings on an African American man with sickle cell disease whose RBCs typed C+c+ and whose plasma contained anti-c. Hemagglutination tests, DNA extraction, PCR-RFLP, reticulocyte RNA isolation, RT-PCR cDNA analyses, cloning, and sequencing were performed by standard procedures. RBCs from the patient typed C+c+ but his plasma contained alloanti-c. DNA analyses showed the presence of RHCE*Ce in trans to RHCE*ceAR with RHD*D and RHD*Weak D type 4.2.2. The amino acid changes on RhceAR are such that C+c+ patient made alloanti-c. This case shows that RhceAR carries a partial c antigen and illustrates the value of DNA testing as an adjunct to hemagglutination to aid in antibody identification in unusual cases.

Molecular bases of unexpressed RHD alleles in Chinese D- persons.

BACKGROUND: The aim of this study was to use a systematic survey to analyze RHD alleles in Chinese D- donors who do not express D antigen or who lack functional RhD protein. STUDY DESIGN AND METHODS: A total of 733 D-Chinese donors, not including Del phenotypes, were investigated by RHD polymorphism­specific polymerase chain reaction (PCR), Rhesus box PCR-PstI digestion, and RHD sequencing. The frequencies of identified alleles were calculated. RESULTS: Three genetic mechanisms and eight alleles were found associated with the Chinese D- phenotype. One new RHD/CE hybrid allele and one novel mutation were also found. The rates of total deletion and the most frequent hybrid allele RHD(1)-CE(2-9)-D(10) were similar to those found in previous studies. A previously reported mutation RHD(711delC) was found to be the predominant cause of aberrant RHD alleles. CONCLUSION: Informative population-based data for improving molecular diagnostic strategies for Chinese D- persons are suggested by this study. This type of systematic knowledge is important for the development of typing and transfusion strategies for the Chinese population.

Molecular basis of the Rh antigen RH48 (JAL).

BACKGROUND AND OBJECTIVES: RH48 (JAL) is a low-incidence Rh antigen of unknown molecular background associated with weakened expression of RhCE antigens. The objective of this study was to establish the molecular basis of JAL. MATERIALS AND METHODS: Seventeen JAL+ samples, from seven black (one of them a Brazilian of mixed race: black/Caucasian), nine European Caucasians and one Asian individuals, were typed with anti-D, -C, -c, -E and -e. Some samples were also tested for V/VS and ce (f). Titration studies and flow cytometry were used to analyse the expression of the JAL antigen and genomic DNA sequencing of all RHCE exons was conducted on all samples. Routine genotyping for RHCE was carried out on all samples. Screening of RHD exons 1-10, which included detection of the DAU allele, was carried out on all except one of the black samples. The Caucasian samples and remaining black sample were screened for the DAU mutation 1136C>T (T379M). RESULTS: Six black individuals had the Dce haplotype with RHCE mutations 340C>T (R114W) and 733C>G (L245V) [V/VS] and the RHD mutation T379M [DAU]. One mixed race individual had the Dce haplotype with the RHCE mutation 340C>T (R114W) but without the V/VS or DAU mutation. Eight Caucasians had the DCe haplotype with the 340C>T mutation. One Caucasian and one Asian had the Dce haplotype with a different mutation in an adjacent nucleotide, 341G>A (R114Q). All Caucasian individuals were negative for the DAU mutation 1136C>T (T379M). Previously described weakness of CE-related Rh antigens when present in single dose on JAL+ samples of DCe and Dce haplotypes was observed. Weak expression of V/VS was observed in the three black samples tested and weakness of JAL was observed in the black samples compared to the Caucasian samples. CONCLUSION: The same mutation (340C>T, R114W) in two different haplotypes (DCe and Dce) and another mutation (341G>A, R114Q) in one of these haplotypes (Dce) are associated with expression of the JAL antigen. One of the RHCE mutations detected in our samples (340C>T) has been previously described but not in association with the JAL antigen. Our results indicate that the previously described RhCeMA and ce(s)(340) alleles encode the JAL antigen. Expression of V/VS antigen is weakened in the presence of JAL and expression of JAL is usually weaker when associated with the Dce haplotype compared to DCe.