by Cheng-Han Huang, Ying Chen, Marion E. Reid, and Christine Seidl Rhnull Disease: The Amorph Type Results From a Novel Double Mutation in RhCe Gene on D-Negative Background by Cheng-Han Huang, Ying Chen, Marion E. Reid, and Christine Seidl Blood Volume 92(2):664-671 July 15, 1998 ©1998 by American Society of Hematology

Gross structure of RH30 locus in the amorph Rhnull family as determined by Sph I RFLPs. (Left) Southern blot analysis of 4 members from the Rhnull family. Gross structure of RH30 locus in the amorph Rhnull family as determined by Sph I RFLPs. (Left) Southern blot analysis of 4 members from the Rhnull family. Lane 1, D-positive; lane 2, D-negative; and lanes 3 through 6, family members II-2, II-3, III-1, and III-2, respectively. Genomic DNAs were digested with Sph I and the polymorphic regions spanning exons 4-7 are shown. Note that RhD gene bands (8.9 and 1.2 kb) are missing in II-2, II-3, and III-2, whereas the RH50 locus is grossly normal in all members (gels not shown). Size marker (in kilobases) of λ phage DNA cleaved by HindIII is indicated. (Right) Family pedigree transmitting a putative amorph Rhnull disease gene. I-1, I-2 (deceased), II-1, and II-4 were not available for molecular analyses. II-2 (propositus) and II-3 are Rhnullhomozygotes, whereas their children, III-1 and III-2, are heterozygotes. The genotype of each member was deduced from RFLP analysis and phenotyping. The amorph copy of RhCe gene on RhD deletion background is denoted by a crossed box with a vertical line. Cheng-Han Huang et al. Blood 1998;92:664-671 ©1998 by American Society of Hematology

Assay of Rh30 exons in the Rhnull family by PCR and restriction analysis. Assay of Rh30 exons in the Rhnull family by PCR and restriction analysis. Individual exons were amplified by PCR and their gene origin was determined by unique restriction site or primer specificity.29 The digested PCR products were separated by native 6% PAGE and stained with ethidium bromide. Enzymes used and exon numbers are indicated above gel panels. Lanes 1 through 6 are as in Fig 1. Below each panel, the size and gene origin of the respective fragments are shown (“+” indicates a cleavage). In the exon 10 panel, the expected RhD (233 bp) and RhCe (163 bp) bands were from coamplification of both gene fragments using a common 5′ primer, Ex-10s, coupled with two 3′-UTa primers (Table 1). Exons 3 and 8 were not analyzed, because the former lacks the unique site and the latter is identical between RhCE and RhD.6-10 Cheng-Han Huang et al. Blood 1998;92:664-671 ©1998 by American Society of Hematology

Identification of the novel mutation in RhCe gene by RT-PCR and sequencing. 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 Rhnull 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). Cheng-Han Huang et al. Blood 1998;92:664-671 ©1998 by American Society of Hematology

The deduced amino acid sequence and topology of amorph RhCe from the 2 Rhnull patients. The deduced amino acid sequence and topology of amorph RhCe from the 2 Rhnull patients. (A) Comparison of the sequence between the normal (residues 251-417) and putative amorph RhCe protein (residues 251-398). The frameshift starts from Pro323 (marked by a star) and the new stretch of 76 amino acids terminates at Gly398 (bold). (B) Topology and organization of the putative amorph protein in the membrane. The amorph protein lacks the last 2 TM domains and does not express any Rh antigens, although it contains C and e antigen-specific polymorphisms, Ser103 and Ala226, on the second and fourth exofacial loops. (C) Immunoblot analysis of Rh30(D) and Rh50 proteins in the Rhnull membrane. The upper panel was probed with antibody LOR-15C9 and the lower one with 2D10. Lane designations are as in Fig 1. Note that no RhD protein was detected in D-negative and Rhnull patients, as expected. In contrast, a significant amount of Rh50 protein was present in the Rhnull cells. Cheng-Han Huang et al. Blood 1998;92:664-671 ©1998 by American Society of Hematology

Family inheritance of the amorph Rhnull gene. Family inheritance of the amorph Rhnull gene. (A) The genomic region encompassing exon 7 of RhCE gene was amplified by 2 pairs of primers In-6s/Ex-7a and In-6s/In-7a (Table 1). The In-6s/Ex-7a fragments were cut by BamHI (G↓GATCC), which is diagnostic of the mutation, and separated by native 6% PAGE. Although no cleavage is seen in control lanes, the respective fragments of 264 bp (denoted by a star) were cut completely in the Rhnullpatient and partially in the heterozygotes. (B) Sequencing of the genomic fragments In-6s/In-7a. The 2 patients showed the same mutation, 2 deleted nucleotides (966T and 968A, boxed), in this fragment, which was sequenced on both strands. Intronic nucleotides are denoted by lowercase letters and exonic ones by uppercase letters. Dashes indicate identical nucleotides. The 2 primers for PCR amplification are overlined. Cheng-Han Huang et al. Blood 1998;92:664-671 ©1998 by American Society of Hematology

Two models accounting for origin of the double mutation in the amorph Rhnull disease gene. Two models accounting for origin of the double mutation in the amorph Rhnull disease gene. (A) Spontaneous mutation model. Sequences of codons 318-327 in RhD and RhCe genes are shown on top. Three mismatches at center right are marked by stars. Possible arrangements of the mutated region (boxed) in the amorph gene are depicted in two hypothetical schemes (see Discussion for details). a and b denote alternatives of the same scheme. Scheme I shows a noncontiguous deletion of two nucleotides, whereas scheme II shows a contiguous deletion of 2 nucleotides in association with a T→C transition. The BamHI site is shown. (B) Microgene conversion model. A heteroduplex is formed between RhD and RhCe genes via homologous pairing and strand synapsis. A failure in repair synthesis involving codons 322 and 323 would result in A→C transition and contiguous deletion of 2 nucleotides (boxed). This model is compatible with scheme II shown above but accommodates the latter in a single event. Cheng-Han Huang et al. Blood 1998;92:664-671 ©1998 by American Society of Hematology