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

MUFARREGE N; TRUCCO BOGGIONE C; PRINCIPI C; LUJÁN BRAJOVICH M; MATTALONI S; BIONDI C; ARNONI C; CASTILHO L; COTORRUELO C

Basilea

Congreso; 29th Regional Congress of the ISBT; 2019

International Society of Transfusion Medicine

Background: Rhnull or Rhmod ?the so-called Rh-deficiency phenotypes- are characterized by a null or severely reduced RH antigen expression (including D, C/c and E/e), respectively. Molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. Rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. The former is caused by homozygosity for silent genes at RHD and RHCE loci, caused by inactivating mutations in RHCE and deletion of RHD. On the other hand, the regulator Rhnull type as well as the Rhmod phenotype are attributed to mutations in RHAG gene when in homozygous state or when in heterozygosity with another RHAG allele containing an inactivating mutation. A functional RhAG is essential both for the correct Rh complex assembly and Rh antigen expression in the erythrocyte membrane. Aims: The aim of this study was to investigate the molecular genetic basis of an Argentinean proband with no detectable D, C, c, E and e antigens by standard serological techniques. 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, increased LDH and marked spherocytosis. The D, C, c, E and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. Genomic DNA was isolated using a modified salting-out method. DNA samples were initially screened for the presence of intron 4 and the 3? untranslated region of the RHD gene using PCR strategies. RHC/c, and RHE/e alleles were studied by allele-specific PCRs to determine the RHCE genotype. RHD zygosity was analyzed by PCR-RFLP. RHD exon polymorphisms were studied by RHD exon scanning procedure based on PCR-SSP. RHAG gene was investigated by exon-specific PCR amplification and 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 Rh phenotype was: D+, C+, c-, E-, e+. RH genotyping confirmed the Rh phenotypes for all 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 exons were detected by exon scanning. Sequencing analysis revealed an homozygous c.920C>T mutation in RHAG exon 6 in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. The c.920C>T mutation is responsible for the p.Ser307Phe amino acid substitution predicted to be in the 10th RhAG glycoprotein transmembrane segment. Summary / Conclusions: This study described the molecular background responsible for an Rh-deficiency phenotype in an Argentinean proband. We identified the novel missense mutation c.920C>T in the RHAG gene which results in the Ser to Phe single amino acid substitution that shows to be critical for Rh antigen complex assembly within the erythrocyte membrane. Further studies are being performed in order to determine whether the proband is Rhnull or Rhmod.