DO/ART4 gene sequencing in sub-Saharan cohorts and African migrants: useful data describing the diversity and spreading of rare variants.

BACKGROUND: Due to the unavailability of immunological reagents, the Dombrock blood group is insufficiently explored in African populations and can be a source of alloimmunization. A large study including pygmoid and nonpygmoid ethnic groups from East, Central, and West continental Africa, together with African migrants like Comorians, Afro-Caribbean from Martinique, and Maroons from French Guiana would be helpful to increase transfusion safety. STUDY DESIGN AND METHODS: Using genomic DNA extracted from blood samples collected from 336 nonpygmoid and 51 pygmoid Africans as well as 268 samples of African descent, DO coding regions were PCR-amplified and sequenced. RESULTS: DO*A and DO*B alleles were detected in almost all groups, with a clear predominance of DO*B in every cohort tested. DO*JO and DO*HY allele frequencies reached 10% or more in several ethnic groups. DO*B-SH-Gln149Lys, DO*B-Ile5Thr, and DO*DODE variants were identified both in African ethnic groups and outside Africa. Twelve novel variants were characterized on a DO*A or a DO*B background. Five of them were found in both African and migrant cohorts, the others were restricted to either within or outside Africa. No DO*DOYA, DO*DOLG, DO*DOLC, nor DO*DOMR variants were observed. A first phylogenetic tree was proposed including all variant alleles. CONCLUSION: This study across continental Africa and countries with African migrants provides a useful overview of Dombrock allele diversity and distribution. The identification of 12 new alleles underlines the importance of genotyping for Dombrock alleles, particularly to improve transfusion safety in countries hosting migrant populations of African descent.

Sequencing of the ART4 gene in sub-Saharan cohorts reveals ethnic differences and two new DO alleles: DO*B-Ile5Thr and DO*B-Trp266Arg.

BACKGROUND: Given the high heterogeneity of sub-Saharan populations especially between nonpygmoids and pygmoids, differences are expected during investigation of the DO/ART4 gene. STUDY DESIGN AND METHODS: Using genomic DNA extracted from blood samples collected from 77 Tswa pygmoids and 39 Teke and seven San nonpygmoids, DO coding regions were amplified and sequenced. A tetra-primer amplification refractory mutation system-polymerase chain reaction method was developed to specifically detect the DO*B-SH-Gln149Lys variant. Membrane expression of newly identified variant alleles in K562-transduced cells was studied by flow cytometry. RESULTS: Extensive polymorphism was confirmed in Teke or San nonpygmoids and Tswa pygmoids with, respectively, 12, zero, and 24 DO*A; 54, 10, and 115 DO*B or DO*B-WL; five, zero, and 14 DO*HY; and six DO*JO alleles in Teke only. The DO*B-SH-Gln149Lys variant was observed as the third most frequent after the DO*HY and DO*JO alleles. Two novel DO*B alleles were identified in the San samples, that is, DO*B-Ile5Thr and DO*B-Trp266Arg. Study of K562-transduced cells showed that compared to the DO*B allele, DO*B-Ile5Thr was expressed more strongly while DO*B-Trp266Arg variant was expressed to a lesser extent and was not recognized by MIMA-123 monoclonal antibodies. CONCLUSION: Sequencing analysis showed more allelic combinations in nonpygmoids than in pygmoids with high frequencies of DO*HY, DO*JO, and DO*B-SH-Gln149Lys variant alleles. This finding underlines the importance of including DO*HY and DO*JO single-nucleotide polymorphisms in genotyping tests to improve transfusion safety. Characterization of two novel DO*B alleles highlights the value of testing selected ethnic groups in understanding DO allele diversity.

Synonymous nucleotide polymorphisms influence Dombrock blood group protein expression in K562 cells.

To gain further insight into ART4 (DO) gene alleles (DO*A, DO*JO1, DO*A-WL, DO*DOYA, DO*B, DO*B-WL, DO*B-SH-Q149K, DO*B-(WL)-I175N, DO*HY1, DO*HY2, DO*DOMR) and evaluate the impact of synonymous nucleotide polymorphisms on protein expression and mRNA accumulation of DO*A-HA, DO*A-SH and DO*B-SH alleles, human erythroleukaemic K562 cells were transducted with variant DO-lentiviral particles and analysed by flow cytometry and quantitative reverse transcription polymerase chain reaction. Monoclonal antibody (MoAb) detection of DO*A-HA and DO*JO1 transductants was lower than DO*A transductants, while detection of DO*A-SH, DO*A-WL and DO*DOYA transductants was higher. Variant DO*B alleles, i.e. DO*B-SH, DO*B-WL, DO*HY1, DO*HY2 and DO*DOMR, showed reduced MoAb binding. The unexpected modifications of protein expression of the DO*A-HA, DO*A-SH and DO*B-SH alleles that differ from the DO*A or DO*B alleles by a single synonymous polymorphism were abolished by reversion, thus implying involvement of these polymorphisms. Depending on the Leu208 codon used, detection level ranged from 1 to 4·14. In the variant alleles resulting from single synonymous polymorphism, mRNA accumulation correlated roughly with MoAbs detection levels, suggesting post-transcriptional regulation. Other than a few reports involving aberrant splicing, the experiments described herein provide the first evidence that synonymous nucleotide polymorphisms can influence Dombrock blood group expression. Such polymorphisms should be taken into account for molecular screening and potential impact on transfusion.

Characterization of novel RHD alleles: relationship between phenotype, genotype, and trimeric architecture.

BACKGROUND: RH1 is one of the most clinically important blood group antigens in the field of transfusion and prevention of fetomaternal incompatibilities. New variant RHD alleles are regularly identified and their characterization is essential to ensuring patient safety. STUDY DESIGN AND METHODS: Blood samples with uncertain RhD phenotypes not resolved by our first-line SNaPshot assay were sequenced for all 10 RHD exons. RHD zygosity was investigated. Flow cytometry was performed to determine RhD antigen density and epitope pattern. RESULTS: Seven novel RHD alleles were identified. Six, that is, RHD(T55P), RHD(A85G), RHD(G132R), RHD(G132E), RHD(D403V), and DAR(T203A), resulted from nucleotide polymorphisms. The seventh, that is, RHD(S182WfsX46), resulted from a 4-bp deletion that led to a reading frame shift and the appearance of a premature stop codon. Study of RhD expression of the first five alleles at hemizygous state showed greatly reduced antigen densities ranging from 50 to 618 antigens per red blood cell (RBC). DAR(T203A) was classified as a partial D antigen with a weakened reactivity profile similar to that of DAR. As expected, no D antigen was detected on RBCs carrying the RHD(S182WfsX46) allele. In parallel, RhD expression of RHD(G336R)/weak D type 58, RHD(F410V), and suspected RHD(1-9)-CE was determined to be less than or equal to 50 antigens per RBC. RhAG/RhD(2) trimer model supports the observed phenotypes. CONCLUSION: Although the frequency of the new RHD alleles presented herein is low, their phenotypic and genotypic description adds to the repertoire of reported RHD alleles. These data can be useful for optimization of molecular screening tools.

Dombrock genotyping in a native Congolese cohort reveals two novel alleles.

BACKGROUND: Since variant alleles in the Dombrock (DO) blood group system are common in Africans, DNA typing of DO alleles in an uninvestigated Congolese Teke ethnic group was performed. STUDY DESIGN AND METHODS: DO exons were polymerase chain reaction amplified, using genomic DNA extracted from blood samples, and sequenced. Membrane expression in K562 cells transduced with DO-cDNAs using lentiviral vectors was studied by flow cytometry. Amino acid changes were mapped on the protein structure, predicted by homology modeling. RESULTS: In 41 samples investigated, there were 56 DOB or DOB-WL (68%), 15 DOA (18%), 6 HY (7%), and 3 JO (4%) alleles. The remaining two alleles were novel, that is, DOB-SH-Gln149Lys carrying a 445C>A transversion and DOB-(WL)-Ile175Asn showing a 524T>A transversion on a DOB or DOB-WL background. Transduced K562 cells revealed that DOB-SHGln149Lys variant was expressed to the same extent as DOB-SH but to a lesser extent than the DOB control. The DOB-Ile175Asn variant shows equivalent expression to DOB but is not recognized by monoclonal antibodies MIMA-53. As deduced from the protein model, these missense changes would lead to structure similar to the wild-type one, with only modified surface features. CONCLUSION: Molecular screening of Teke individuals revealed a high frequency of HY and JO alleles and two novel alleles, one on the DOB (or DOB-WL) and one on the DOB-SH background. Expression studies highlighted the impact of changes on Do protein expression. These findings suggest that allelic diversity is greater than expected and that expression level of DO alleles should be taken into account in transfusion

Molecular analysis of inactive and active RHD alleles in native Congolese cohorts.

BACKGROUND: In Africa, RHD alleles have not been fully characterized. The purpose of this study was to identify inactive and active RHD alleles at the molecular level in Congolese cohorts. STUDY DESIGN AND METHODS: Blood samples were collected from people living in central Congo populated by Teke ethnic group. A total of 110 D- and 40 D+ samples from Congo-Brazzaville and Teke groups, respectively, were selected for RHD genotyping using allele-specific primer polymerase chain reaction and sequencing. RESULTS: In the 110 D- samples, RHD exon amplifications were observed in 7 samples that were subsequently identified by sequencing as weak D type 4 variants. In the remaining 103 D- samples, the frequencies of RHD gene deletion, RHDpsi pseudogene, and RHD-CE-D(s) hybrid gene were 0.75685, 0.20560, and 0.04468, respectively. In the D+ samples, 26 individuals carried at least a regular RHD gene; 9 carried aberrant RHD alleles belonging to the African D clusters, that is, DAU, DIVa, and weak D type 4; 3 carried RHDpsi in trans with a DAU allele including one novel RHD allele (V279M, S333N, T379M) named DAU-7; and 2 others were partially determined. CONCLUSION: This study revealed a high frequency of weak D type 4 alleles that confirmed the need to use indirect antiglobulin test to improve transfusion safety in the Congo and in countries hosting Congolese people. Findings also indicated that there is a geographic variation in RHD allele distribution and showed that RHD gene deletion is the most prevalent cause of the D- phenotype in the Congolese population.

Using an EGFPmeter to evaluate the lentiviral vector production: tricks and traps.

An efficient viral vector containing supernatant is the first key issue to address for gene therapy or basic research projects relying on viral gene transfer. When present in a lentiviral vector backbone as a reporter gene, the expression of the enhanced green fluorescent protein (EGFP) shows strong interests to assess the production of lentiviral vector supernatants. The immediate nondestructive visual analysis of EGFP expression helps to predict the quality of the viral vector supernatant while it is produced. It also largely facilitates the quantitative evaluation of the number of viral particles present in the collected supernatants. Although some traps should be considered when analyzing the expression of a lentivirally transduced EGFP expression unit, the EGFP is the most powerful tool to consider when designing new gene transfer and expression strategies.