Novel missense mutation in RHAG gene causes the first reported Rh-deficiency phenotype in Argentina

BARTOLI, SONIA; LUJÁN BRAJOVICH, MELINA ELIANA; CRUZ, I; CASTILHO, LILIAN; FRANCO, NOELIA; PRINCIPI, CINTIA; SEVERICH, I; ARNONI, CARINE; MUFARREGE, NICOLÁS; TRUCCO BOGGIONE, CAROLINA; MATTALONI, STELLA MARIS; BIONDI, CLAUDIA; COTORRUELO, CARLOS

Basilea

Congreso; 29th Regional Congress of the ISBT; 2019

ISBT

Background: Rhnull or Rhmod ?the so-called Rh-deficiency phenotypes- are characterizedby a null or severely reduced RH antigen expression (including D, C/c and E/e), respectively. Molecular genetic studies showed that these phenotypes are transmittedin an autosomal recessive manner. Rhnull phenotype originates from two differentmolecular events giving rise to the amorph type and the regulator type. Theformer is caused by homozygosity for silent genes at RHD and RHCE loci, caused byinactivating mutations in RHCE and deletion of RHD. On the other hand, the regulatorRhnull type as well as the Rhmod phenotype are attributed to mutations in RHAG gene when in homozygous state or when in heterozygosity with another RHAGallele containing an inactivating mutation. A functional RhAG is essential both forthe correct Rh complex assembly and Rh antigen expression in the erythrocytemembrane.Aims: The aim of this study was to investigate the molecular genetic basis of anArgentinean proband with no detectable D, C, c, E and e antigens by standard serologicaltechniques.Methods: Blood samples were collected from the proband, her parents and sister.The proband was a 14 year-old young woman with parameters of hemolytic anemia:low hemoglobin level (10 g/dL), reticulocytosis (14%), hyperbilirubinemia, increasedLDH and marked spherocytosis. The D, C, c, E and e status was determined by standardserologic hemagglutination techniques using specific monoclonal antibodies.Genomic DNA was isolated using a modified salting-out method. DNA samples wereinitially screened for the presence of intron 4 and the 30 untranslated region of theRHD gene using PCR strategies. RHC/c, and RHE/e alleles were studied by allele-specificPCRs to determine the RHCE genotype. RHD zygosity was analyzed by PCRRFLP.RHD exon polymorphisms were studied by RHD exon scanning procedurebased on PCR-SSP. RHAG gene was investigated by exon-specific PCR amplificationand Sanger sequencing.Results: No D, C, c, E and e antigens were detected in the proband?s erythrocytes.The father and sister Rh phenotype was: D+, C+, c+, E+, e+ whereas the mother Rhphenotype was: D+, C+, c-, E-, e+. RH genotyping confirmed the Rh phenotypes forall family members except for the proposita who genotyped RHD+, RHC+ and RHe+.All samples showed an homozygous status for the RHD gene and all RHD exonswere detected by exon scanning. Sequencing analysis revealed an homozygousc.920C>T mutation in RHAG exon 6 in the proband whereas the rest of the familyshowed an heterozygous state in the same nucleotide position. The c.920C>T mutationis responsible for the p.Ser307Phe amino acid substitution predicted to be inthe 10th RhAG glycoprotein transmembrane segment.Summary/Conclusions: This study described the molecular background responsiblefor an Rh-deficiency phenotype in an Argentinean proband. We identified the novelmissense mutation c.920C>T in the RHAG gene which results in the Ser to Phe singleamino acid substitution that shows to be critical for Rh antigen complex assemblywithin the erythrocyte membrane. Further studies are being performed in orderto determine whether the proband is Rhnull or Rhmod.