Yann Fichou1, Cédric Le Maréchal, Laurence Bryckaert, Isabelle Dupont, Déborah Jamet, Jian-Min Chen, Claude Férec. 1. Etablissement Français du Sang (EFS)-Bretagne, Brest, France; Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France; Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France; Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Régional Universitaire (CHRU), Hôpital Morvan, Brest, France.
Abstract
BACKGROUND: Molecular biology techniques, such as single specific-primer polymerase chain reaction (PCR), denaturing-high performance liquid chromatography, direct sequencing, next-generation sequencing, and microarray platforms, contribute to the efficient genotyping of the human blood group RHD gene. However, some alleles remain undetermined in rare cases in DNA samples carrying two copies of the RHD gene, which challenge the identification of D-CE hybrid genes. STUDY DESIGN AND METHODS: We set up, in a single-tube format, a qualitative and quantitative assay based on multiplex PCR of short fluorescent fragments (QMPSF) to simultaneously amplify all 10 RHD exons on the one hand and all 10 RHCE exons on the other hand. RESULTS: The test proved to be useful to rapidly identify hybrid genes in hemizygous RHD samples carrying a hybrid D-CE gene and to resolve unknown genotypes by quantifying individual exons in compound heterozygous samples, but also unexpectedly helped to redefine the RHDΨ haplotype. While validating the test, two novel single-point variants, c.648G>C (p.L216F) and c.1048G>C (p.D350H), were found. CONCLUSION: For the first time, a QMPSF-based method is reliable to individually quantify the exons of both RH genes, including hybrid D-CE genes in compound heterozygous samples and may help to investigate samples with unknown RHD and/or RHCE status.
BACKGROUND: Molecular biology techniques, such as single specific-primer polymerase chain reaction (PCR), denaturing-high performance liquid chromatography, direct sequencing, next-generation sequencing, and microarray platforms, contribute to the efficient genotyping of the human blood group RHD gene. However, some alleles remain undetermined in rare cases in DNA samples carrying two copies of the RHD gene, which challenge the identification of D-CE hybrid genes. STUDY DESIGN AND METHODS: We set up, in a single-tube format, a qualitative and quantitative assay based on multiplex PCR of short fluorescent fragments (QMPSF) to simultaneously amplify all 10 RHD exons on the one hand and all 10 RHCE exons on the other hand. RESULTS: The test proved to be useful to rapidly identify hybrid genes in hemizygous RHD samples carrying a hybrid D-CE gene and to resolve unknown genotypes by quantifying individual exons in compound heterozygous samples, but also unexpectedly helped to redefine the RHDΨ haplotype. While validating the test, two novel single-point variants, c.648G>C (p.L216F) and c.1048G>C (p.D350H), were found. CONCLUSION: For the first time, a QMPSF-based method is reliable to individually quantify the exons of both RH genes, including hybrid D-CE genes in compound heterozygous samples and may help to investigate samples with unknown RHD and/or RHCE status.