Frequency and characterization of RHD and RHCE variants in the Noir Marron population from French Guiana.

BACKGROUND: The RH system is one of the most polymorphic blood group systems due to the proximity and opposite orientation of RHD and RHCE genes. Numerous alleles are described and can affect Rh protein expression. This complexity is especially evident in populations of African origin. We performed RHD and RHCE genotyping of the Noir Marron population in French Guiana. This population belongs to the Maroon community who are direct descendants of African slaves, who escaped from Dutch plantations, in the current day Suriname, during the 17th century. They represent an original ethnic group with highly blended culture. METHODS AND MATERIALS: A total of 89 DNA samples were collected from four different ethnic groups of the Noir Marron population of French Guiana. RHD and RHCE genotyping was performed using DNA microarray and/or sequencing. RESULTS AND DISCUSSION: Significant allelic diversity was shown, with 45% of individuals presenting an RHD gene variant (most common: RHD*DAU, RHD*DIVa, and RHD*DIIIa allele) and 9.4% with a partial D phenotype. Likewise, 85% presenting an RHCE gene variant and 9% a partial RH2 antigen. One original allele was identified in two D+ Noir Marron individuals: a hybrid RHD*DIIIa-CE(9)-D allele, encoding probably a partial D antigen and associated with an RHCE*ce(48C,733G,1006T) allele. The African diversity of RHD and RHCE genes is found in this population with preserved genetic but mixed cultural backgrounds. These data allow us to describe the characteristics of the RH system antigen and highlights a significant number of partial antigens with a risk of alloimmunization.

Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis.

BACKGROUND: Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®). RESULTS: Statistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient. CONCLUSIONS: This comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.

New KEL*01M and KEL*02M alleles: structural modeling to assess the impact of amino acid changes.

BACKGROUND: The KELL antigens are carried by the well-folded and highly polymorphic glycoprotein KELL, belonging to the M13 family of metalloproteases. Anti-KEL, particularly anti-KEL1, are clinically significant. We retrospectively investigated genomic DNA from samples with uncertain KEL1 or KEL2 phenotype and identified six novel Kmod alleles. We then considered a model of the protein three-dimensional (3D) structure to assess the impacts of the amino acid changes. STUDY DESIGN AND METHODS: The 19 exons of the KEL gene were polymerase chain reaction amplified and sequenced. Modeling was performed using the experimental 3D structure of human endothelin-converting enzyme-1 in the presence of the metabolite phosphoramidon. RESULTS: We identified four novel KEL*01M alleles with amino acid substitutions p.Arg447Trp, p.Gly641Arg, p.Ala645Val, and p.Gly703Arg found buried within helices of the ectodomain catalytic lobe. We also revealed one new KEL*02M allele with p.Gly263Glu in contact with solvent (water) located within the second lobe of the ectodomain. One sample with c.575G>C transversion (p.Arg192Pro) on a KEL*02 background showed a weakened reactivity for KEL1. According to our 3D modeling, these amino acid substitutions may have a profound impact on the protein structure. CONCLUSION: This study is especially interesting with regard to the description of four new KEL*01M alleles. Indeed, to date only two KEL*01M alleles have been described and our data suggest a nonnegligible incidence of KEL1 variants. Serologic KEL2-negative results as well as any ambiguity implying either KEL1 or KEL2 in donors should always be confirmed by means of genotyping analysis and discrepancies between these methods require sequencing of KEL gene.

Five-Years Review of RHCE Alleles Detected after Weak and/or Discrepant C Results in Southern France.

Immunohematology laboratories are regularly facing transfusion issues due to serological weaknesses. Altered (partial) RH antigens account for most of them. In some situations, RHCE variant alleles are involved. Herein we present our three-step molecular exploration, with allele frequencies, that has efficiently untangled RH2 phenotype weaknesses and discrepancies in our 2017-2021 cohort. In the last 5 years, the PACA Corse EFS molecular platform received 265 samples from healthy blood donors or patients with C and C/e typing difficulties. The first-intention technique (DNA array and real time PCR for RHCE*CeRN research) detected RHCE variant alleles in 143 cases (54%). The RHCE alleles classically found in African populations were the most frequent, with RHCE*CeRN allele in 40 cases (15%) and (C)ces haplotype type 1 and 2 in 26 cases (10%). A "CE" effect haplotype was suspected in 56 cases, due to the uncommon DCE haplotype that may explain the low C expression. When there were no RHCE*Ce or RHCE*CE alleles, we then searched for RHD polymorphisms by DNA array. We detected the RHD*DAU5 and RHD*DIVa in 18 and 7 cases respectively, suggesting that C ambiguity is related to the presence of these alleles which has never been described with DAU5. If no variant RHCE and RHD alleles were detected, we finally sequenced the 10 exons of both RHCE and RHD genes according to the clinical context and found seven new RHCE alleles. Thus, this molecular strategy would improve the knowledge of RHCE variants' expression and, thus, optimize the transfusion management.