First report of Rhnull individuals in the Indian population and characterization of the underlying molecular mechanisms.

Abstract

Rhnull phenotype is an extremely rare condition characterized by no expression of Rh antigens at the surface of red blood cells. Although rare, genetic bases of this phenotype are well known and include mutations within either the RH (RHD and RHCE) genes or the RHAG gene. So far Rhnull has been reported in individuals of Caucasian, African, and Asian origin. Here, we report individuals from two families of Indian origin representing such a rare phenotype. Serologic analysis was carried out by testing with anti-D, -C, -c, -E, and -e in Rhnull individuals and their family members. RH genes were analyzed by standard molecular approaches, including Sanger sequencing and quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments. RHAG gene was investigated by exon-specific PCR amplification and Sanger sequencing. In one family, RHAG gene was found to be deleted at the homozygous state in the propositus, suggesting Rhnull of the regulator type. In the other family, a novel splice site variant in RHCE in cis with whole RHD gene deletion was identified at the homozygous state. Further functional analysis by minigene splicing assay showed that this variation, that is, c.801 + 1G>A, completely impairs normal splicing, then inactivating the expression of RhCE protein. Contrary to the former case, these data suggest Rhnull of the amorph type. Overall, we report for the first time the molecular mechanisms responsible for Rhnull phenotype in individuals of Indian origin. This study contributes to extend the molecular spectrum of variations in Rhnull individuals.