Rhnull disease: the amorph type results from a novel double mutation in RhCe gene on D-negative background

Rhnull disease, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic hemolytic anemia. We studied here a German family transmitting a putative amorph Rhnull disease gene and identified a rare mutation causing the loss-of-function phenotype. We analyzed the genomic and transcript structure of RH30, RH50, and CD47, the three loci thought to be most critical for expression of the Rh complex in the red blood cell membrane. We showed that in this family the Rh50 and CD47 transcripts were normal in primary sequence. However, the RH30 locus contained an unusual double mutation in exon 7 of the RhCe gene, in addition to a deletion of the RhD gene. The mutation targeted two adjacent codons in multiple arrangements probably via the mechanism of microgene conversion. One scheme entails a noncontiguous deletion of two nucleotides, [ATT(Ile322)-->AT] and [CAC(His323)-->CC], whereas the other involves a T-->C transition [ATT(Ile322)--> ATC] and a dinucleotide deletion [CAC(His323)-->C]. They caused the same shift in open reading frame predicted to encode a shortened protein with 398 amino acids. The loss of two transmembrane domains and gain of a new C-terminal sequence are likely to alter the protein conformation and impair the Rh complex assembly. Our findings establish the molecular identity of an amorph Rhnull disease gene, showing that Rh30 and Rh50 are both essential for the functioning of the Rh structures as a multisubunit complex in the plasma membrane.