Abstract
Several hundred variant alleles have been reported within the homologous RHD and RHCE genes that encode the antigens involved in the human Rh blood group system, which is of the main interest in the field of both transfusion and obstetrical medicine. Although these variants can be mostly characterized at the molecular level by sequence-specific primer polymerase chain reaction (SSP-PCR) and/or direct sequencing, some allelic combinations remain unresolved by conventional methods. Typically exon deletion or hybrid genes may be difficult to assess in a heterozygous context. Here we describe a qualitative and quantitative method to resolve copy number variations in the RH gene exons by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF).
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References
Daniels G (2013) Rh and RHAG blood group systems. In: Daniels G (ed) Human blood groups, 3rd edn. Wiley-Blackwell, Chichester, pp 182–258
Klein HG, Anstee DJ (2005) The Rh blood group system (and LW). In: Klein HG, Anstee DJ (eds) Mollison’s blood transfusion in clinical medicine, 11th edn. Blackwell Publishing, Oxford, pp 163–208
Avent ND, Ridgwell K, Tanner MJ et al (1990) cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen expression. Biochem J 271:821–825
Chérif-Zahar B, Bloy C, Le Van Kim C et al (1990) Molecular cloning and protein structure of a human blood group Rh polypeptide. Proc Natl Acad Sci U S A 87:6243–6247
Colin Y, Chérif-Zahar B, Le Van Kim C et al (1991) Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood 78:2747–2752
Le Van Kim C, Mouro I, Chérif-Zahar B et al (1992) Molecular cloning and primary structure of the human blood group RhD polypeptide. Proc Natl Acad Sci U S A 89:10925–10929
Chérif-Zahar B, Mattéi MG, Le Van Kim C et al (1991) Localization of the human Rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization. Hum Genet 86:398–400
Arce MA, Thompson ES, Wagner S et al (1993) Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhD-negative individuals. Blood 82:651–655
Mouro I, Colin Y, Chérif-Zahar B et al (1993) Molecular genetic basis of the human Rhesus blood group system. Nat Genet 5:62–65
Flegel WA (2011) Molecular genetics and clinical applications for RH. Transfus Apher Sci 44:81–91
Wang D, Lane C, Quillen K (2010) Prevalence of RhD variants, confirmed by molecular genotyping, in a multiethnic prenatal population. Am J Clin Pathol 134:438–442
Silvy M, Simon S, Gouvitsos J et al (2011) Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles. Transfusion 51:401–411
Fichou Y, Le Maréchal C, Jamet D et al (2013) Establishment of a medium-throughput approach for the genotyping of RHD variants and report of nine novel rare alleles. Transfusion 53:1821–1828
Gassner C, Meyer S, Frey BM et al (2013) Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry-based blood group genotyping–the alternative approach. Transfus Med Rev 27:2–9
Granier T, Beley S, Chiaroni J et al (2013) A comprehensive survey of both RHD and RHCE allele frequencies in sub-Saharan Africa. Transfusion 53(Suppl 2):3009–3017
Kappler-Gratias S, Auxerre C, Dubeaux I et al (2013) Systematic RH genotyping and variant identification in French donors of African origin. Blood Transfus 17:1–8
Maaskant-van Wijk PA, Faas BH et al (1998) Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons. Transfusion 38:1015–1021
Fichou Y, Le Maréchal C, Bryckaert L et al (2013) A convenient qualitative and quantitative method to investigate RHD-RHCE hybrid genes. Transfusion 53(Suppl 2):2974–2982
Haer-Wigman L, Veldhuisen B, Jonkers R et al (2012) RHD and RHCE variant and zygosity genotyping via multiplex ligation-dependent probe amplification. Transfusion 53:1559–1574
Shimizu M, Kosaka N, Shimada T et al (2002) Universal fluorescent labeling (UFL) method for automated microsatellite analysis. DNA Res 9:173–178
Guo DC, Milewicz DM (2003) Methodology for using a universal primer to label amplified DNA segments for molecular analysis. Biotechnol Lett 25:2079–2083
Fichou Y, Chen JM, Le Maréchal C et al (2012) Weak D caused by a founder deletion in the RHD gene. Transfusion 52:2348–2355
Fichou Y, Génin E, Le Maréchal C et al (2008) Estimating the age of CFTR mutations predominantly found in Brittany (Western France). J Cyst Fibros 7:168–173
Casilli F, Di Rocco ZC, Gad S et al (2002) Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments. Hum Mutat 20:218–226
Acknowledgements
This work was supported by the Institut National de la Santé et de la Recherche Médicale (Inserm) et l’Etablissement Français du Sang (EFS)—Bretagne.
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Fichou, Y., Férec, C. (2015). Molecular RHD-RHCE Analysis by Multiplex PCR of Short Fluorescent Fragments. In: Bugert, P. (eds) Molecular Typing of Blood Cell Antigens. Methods in Molecular Biology, vol 1310. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2690-9_8
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DOI: https://doi.org/10.1007/978-1-4939-2690-9_8
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2689-3
Online ISBN: 978-1-4939-2690-9
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