[Identification of nine novel alternative splicing isoforms of RHD mRNA]

To investigate the gene structures of RHD mRNA alternative splicing isoforms, total RNA was extracted from normal cord blood samples and RHD mRNA were detected using reverse transcription and polymerase chain reaction (RT-PCR) method. The PCR products were cloned and directly sequenced. The splicing signals of various isoforms were also analyzed by sequencing of exon-intron boundaries using RHD gene specific primers, and the expressed sequence tags (ESTs) databank was screened to find out other RHD alternative splicing isoforms. Nine novel and three recurrent RHD alternative splicing isoforms were identified, among which six isoforms were RHD-RHCE hybrid genes. Three types of alternative splicing were found including exon skipping, alternative 5' splice sites and alternative 3' splice sites. Exons 2 to 9 were involved in RHD mRNA alternative splicing. Two other isoforms of intron retention type were also found in human ESTs databank. Thus, the mRNA splicing mechanism of RHD gene was very complex, and nine novel alternative splicing isoforms were identified including several isoforms based on homologous RHD-RHCE hybrid gene.