Identification of the novel mutation in RhCe gene by RT-PCR and sequencing. (A) Strategy for synthesis and amplification of Rh30 cDNA. Rh30 mRNA was reverse-transcribed into cDNA with either the gene-specific 3′-UTa primer or (dT)₁₆ oligomer and then amplified with two pairs of upstream primers (Table 1). The cDNA products for Rh30 (left panel) and Rh50 (right panel) were separated on 1.8% agarose gels. Designations 1 through 6 are as in Fig 1. Lane a, 5′-UT/Ex-5a segment; lane b, Ex-4s/3′-UTa segment. Bands at bottom are primer dimers. (B) Nucleotide sequencing profiles for the novel double mutation identified in the amorph form of RhCe cDNAs from the 2 Rh_null patients (B.K. and D.R.). The mutation affects 2 codons (322 and 323) in exon 7, involving 2 single nucleotide deletions, ATT→AT (nt 965 or 966) and CAC→CC (nt 968) (indicated by arrow).