Rh_null Disease: The Amorph Type Results From a Novel Double Mutation in RhCe Gene on D-Negative Background

Rh DEFICIENCY SYNDROME is a rare genetic disorder of the red blood cell (RBC) transmitted via an autosomal recessive mode generally through consanguineous pedigrees.1Its occurrence is characterized by two phenotypic conditions: the absence of all Rh antigens defines Rh_null disease, whereas an extremely suppressed expression defines the Rh_modphenotype. Both Rh_null and Rh_mod patients manifest a mild to moderate hemolytic anemia, and their RBCs show changes in morphology (stomatocytosis) and abnormalities in plasma membranes.2-5 Classic genetic studies suggest that Rh_null disease results from distinct mechanisms, depending on whether the mutations target the Rh antigen locus itself (amorph type) or some unlinked loci that modulate the Rh protein expression (regulator type).1 Biochemical analyses show that Rh_null cells not only lack the carrier antigens but are deficient in several membrane glycoproteins, including Rh50, CD47, LW, and GPB.2-5 This intricate relationship highlights the organization of Rh and its related proteins as a multisubunit complex in the RBC membrane and pinpoints the impaired protein-protein interaction as the hallmark of Rh deficiency syndrome.

Although multiple components are likely involved, the nonglycosylated Rh30 polypeptides (ie, RhD and RhCE) and Rh50 glycoprotein emerge as the most important ones in formation of the Rh complex.3-5Despite the localization of their loci to separate chromosomes, Rh30 and Rh50 belong to the same family and share a notable sequence homology, particularly in their putative α-helices that characterize a 12-transmembrane (TM) topology.6-11 Moreover, evidence is accumulating that Rh30 and Rh50 may interact directly with each other to form a structural core in the RBC membrane.12-14 The other potentially crucial component of the Rh complex may be CD47, an integrin-associated protein,15,16 whose expression is profoundly reduced in Rh_null cells,17,18regardless of the genetic mechanisms involved. The still others, namely LW and GPB, are likely to be dispensable,19,20 because their absence neither disrupts the Rh complex nor alters cell morphology and physiology.

The recent identification of mutations associated with Rh50 gene in regulator Rh_null patients defines the Rh50 glycoprotein as a critical coexpressor of Rh30 polypeptides.21,22Nevertheless, no mutations have been reported to date that target either the RH30 or CD47 locus in Rh_nullpatients.

We report here the identification of the first molecular defect that occurs as an amorph Rh_null disease gene in a German family described 25 years ago.23 We show that in this family the genomic structure and transcript expression of RH50 andCD47 are apparently normal. However, the RH30 locus contains a novel noncontiguous deletion of two nucleotides in exon 7 of RhCe gene, in addition to a complete deletion of RhD gene. This novel double mutation has caused frameshift and premature chain termination, thereby leading to a loss-of-function phenotype.

Control blood samples were from D-positive (genotype, DCe/DCe) and D-negative (genotype, dce/dce) human blood donors. Blood samples under study were from 4 members of the Rh_nullfamily, including 2 homozygotes (II-2, B.K., and II-3, D.R.) and 2 heterozygotes (III-1 and III-2; Fig 1). Members I-1 and I-2 are related23 but not available for molecular analysis. Retesting of the patients' RBCs confirmed the absence of all Rh antigens. Immunoblot of RBC membrane proteins was performed as described.24 Two antibodies, LOR-15C9 for RhD25 and 2D10 for Rh50,26 were used. Peroxidase-conjugated antihuman and antimouse Igs were used as the respective secondary antibodies for band visualization.

RNA was isolated from hemolysates and genomic DNA from leukocytes, as described.27,28 The Rh cDNA probes were as described²⁹; they span the 5′ (exon 1-3), middle (exon 4-7), and 3′ (exon 8-10 plus 3′-untranslated or UT) regions, respectively. The Rh50 and CD47 cDNA probes were as detailed previously.21 Southern blot was performed as described.28 

The expression of transcripts was analyzed by RT-PCR, as described.21,29 Rh30-specific primers are shown (Table 1), and those for Rh50 and CD47 were detailed in our previous report.21 The mRNA was converted into cDNA using either a gene-specific 3′-UTa21 or anchored (dT)₁₆ oligomer.30 Total RNA (2 μg) and 50 ng of primer were incubated at 65°C for 5 minutes and on ice for 5 minutes. Reverse transcription was incubated at 42°C for 75 minutes and inactivated at 72°C for 10 minutes. The cDNA was then amplified by different pairs of upstream primers spanning the entire coding region using AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). PCR was repeated 35 cycles as follows: 94°C for 1 minute, 55°C for 45 seconds, and 72°C for 1 minute. The last step for chain extension at 72°C was for 7 to 10 minutes.

Sequences of Rh30 genes were amplified by PCR using total genomic DNA as a template. Exon sequences were analyzed by diagnostic restriction enzymes to distinguish their D or CEorigin.29 To retrieve unknown intron sequences for primer design (Table 1), genomic libraries generated by 6 restriction enzymes were amplified with Rh30 exon-specific primers, as described.21 Amplification was repeated 30 cycles as follows: 94°C for 1 minute, 60°C for 30 seconds, and 72°C for 30 seconds.

Amplified cDNA and genomic products were purified by native 5% polyacrylamide gel electrophoresis (PAGE) and sequenced in both strands on a Model 373A automated sequencer (Applied Biosystems, Foster City, CA). The nucleotide and deduced amino acid sequences were analyzed using the DNASIS program (Hitachi, South San Francisco, CA).

Figure 1 shows a representative Sph1 blot of the Rh_null family hybridized with the cDNA probe spanning the polymorphic region of RH30.31 Notably, the homozygotes and one heterozygote each had a single 7.3-kb band retaining exons 4-7 whose intensity was comparable to that of D-negatives. This indicated that the Rh_null patients lacked RhD gene but had two copies of RhCE gene (Fig 1, left). The other heterozygote inherited from her father a DCe haplotype; thus, her D-specific (8.9 and 1.2 kb) and Ce-specific (5.4 and 1.9 kb) bands showed reduced intensity. Analysis of RH50and CD47 showed no difference between controls and Rh_null family members (gels not shown), indicating that all family members had two intact copies of Rh50 and CD47 genes. These results indicate that the amorph gene occurs on background ofRHD deletion and that both copies of RHCE are defective in the Rh_null patients (Fig 1, right).

To determine whether the RhD gene deletion is total or partial, we analyzed 8 of the 10 exons of Rh30 genes by PCR and diagnostic restriction cleavage (Fig 2). Whereas heterozygote III-1 had a pattern apparently identical with that of D-positives, other members lacked D-specific exons 1, 4, 5, 6, 7, 9, and 10. With regard to the cleavage of exons 1 and 2, the two homozygotes contained Ce- or CE-specific exons, whereas the heterozygotes exhibited a composite pattern. Taken together, these data demonstrate a total absence of RhD gene in Rh_nullpatients and indicate that some subtle molecular defect(s) have silenced the phenotypic expression of their RhCE gene.

Although being a consanguineous pedigree,23 the inheritance pattern of this Rh_null family (Fig 1) did not rule out the occurrence of possible mutations in related suppressor loci. Therefore, we analyzed and sequenced the Rh50 and CD47 cDNAs from the Rh_null patients. No abnormality was found in the Rh50 (Fig 3A) or CD47 transcript (not shown), conforming to the transmission of an amorph gene.

To define the nature of the amorph mutation, the Rh30 cDNA was amplified with different pairs of gene-specific primers. In both Rh_null patients, only RhCE but not RhD cDNA was detected, whose size and amount were comparable to that of controls (Fig 3A). Sequencing showed that the Rh30 cDNA was of Ce type, containing C- and e-specific polymorphisms.6,7,10 However, this cDNA harbored a very rare double mutation in exon 7, which targeted two adjacent codons (322 and 323) and caused a noncontiguous deletion of two nucleotides, 966T and 968A (Fig 3B). Significantly, the mutation resulted in both frameshift and premature termination. Thus, the cDNA was predicted to encode a shortened RhCe-like polypeptide of 398 amino acids (Fig 4A). The premature termination ablated the last 2 TM domains of the protein, whereas the frameshift gave rise to a new C-terminal sequence of 76 amino acids likely facing the cytoplasmic side (Fig 4B). We did not try to establish the status of the RhCe-like protein in the membrane, because no antibody can detect RhCE proteins by immunoblot. However, we showed an absence of RhD but a significant expression of Rh50 in the Rh_nullpatients (Fig 4C). In light of a normal structure of Rh50 and CD47, we conclude that the double deletion is the primary defect underlying the amorph Rh_null disease gene in this family.

The detection of a unique RhCe-like transcript (Fig 3) indicated that the patients are homozygous, whereas their children are heterozygous, for the mutation. As a new BamHI site was created, we performed a diagnostic assay in the 4 members. Whereas no cleavage was seen in controls, a total digestion in the 2 patients and a partial digestion in their children were observed (Fig 5A). This pattern is entirely consistent with the transmission of the amorph gene in an autosomal recessive fashion. Direct sequencing of the exon 7 containing fragment showed the same noncontiguous deletion (Fig 3B), confirming homozygosity of the mutation in the 2 patients.

In this study, we examined a German family in which a putative amorph Rh_null disease gene is transmitted on a consanguineous background.23 Molecular analysis of three genetic loci relating to the expression of the Rh complex, RH30, RH50, andCD47, has led to the detection of a rare double mutation in the Rh30 gene and exclusion of possible candidate mutations in Rh50 and CD47 genes. The mutation is defined as a noncontiguous deletion of 2 nucleotides targeting 2 adjacent codons in exon 7 of the RhCe gene and thus becomes the first known amorph defect at RH30. With a deleted RhD gene positioned in cis and trans, the mutation has silenced the antigen expression of the remaining RhCe gene, causing a loss-of-function phenotype in the two Rh_null patients.

Of the double deletions identified, one hit the second or third position of codon ATT(Ile₃₂₂) and the other the second position of codon CAC(His₃₂₃). Based on the alignment of deletion forms, two models may be postulated to account for their origin. In spontaneous model, two hypothetical schemes could be proposed, each evoking two molecular events. In scheme I, the deletion of 966T (or 965T) and 968A is assumed to occur separately (Fig 6A). In scheme II, one event causes a transition [ATT→ATC] and the other a dinucleotide deletion [CAC→C] (Fig 6A). In the microgene conversion model,32the same end product may arise by a single event in which a heteroduplex of RhD and RhCe could be formed via homologous pairing; failure in repair synthesis involving codons 322 and 323 would result in patched transfer of the RhD-specific C residue and a contiguous deletion of TC dinucleotide (Fig 6B). Although microgene conversion might be infrequent in the Rh system,33 it is implicated as an important mechanism for the diversity of Ig and MHC gene families and provides a plausible explanation to the origin of some rare spontaneous mutations.34,35 

The amorph mutation has targeted a nonconserved region, possibly the fifth endofacial loop, in the Rh30 polypeptides.6-10 Thus, its result as a loss-of-function phenotype is most likely due to the inevitable shift in open reading frame. The status of the putative RhCe-like protein in the Rh_null cells remains to be established. Nevertheless, several lines of evidence suggest that the mutant protein may be inserted into the membrane but not accessible to the antibodies that are strictly conformation-dependent. (1) The RhCe-like transcript was abundantly expressed, suggesting that the steady-state level of mRNA is not affected by the mutation. (2) Albeit at a reduced level, Rh50 was readily detectable by immunoblot. As a critical interaction partner of Rh30,12-14 this expression implies a possible association of Rh50 with the RhCe-like protein. (3) Prior surface-labeling studies detected a structurally similar Rh30 polypeptide in the Rh_null membrane,36indicating that the absence of Rh antigens does not necessarily mean the absence of Rh proteins. As predicted, the primary sequence of the RhCe-like protein indicates a loss of 2 TM domains and a gain of 76 new amino acids in the C-terminal region. Such structural changes would disturb the conformation of the RhCe-like protein and affect its interaction with Rh50 and other related glycoproteins in the RBC membrane.

Identification of the first amorph Rh30 gene establishes that Rh30, akin to its interacting partner Rh50, is an essential member of the Rh membrane structures. However, the molecular details regarding this protein-protein interaction remain to be elucidated. Limited proteolysis and antibody probing suggested that Rh30 and Rh50 are each composed of two 6-TM domains, forming a tetramer through N-terminal contacts.13 However, additional contact sites are likely present to further dictate the assembly of Rh membrane structures. The amorph protein has a change in the C-terminal portion after Pro₃₂₃, but its N-terminal sequence is identical with the wild-type one. Its association with the dysfunction of the Rh complex suggests a possible interaction of Rh30 with Rh50 through their C-terminal regions. Notably, the C-termini of Rh50 mutants in regulator Rh_null disease resulting from a splicing donor mutation21 or a point deletion22 are also abnormal because of the inherent frameshift and premature termination. In the former, the only difference from the Rh50 protein lies C-terminal to the 10th TM domain, whereas the latter is altered after the 11th TM domain. Missense mutations targeting the conserved C-terminal TM domains of Rh50 are also associated with regulator Rh_null (our unpublished data). These coincidental observations suggest that the C-terminal regions of Rh30 and Rh50 play a crucial role in forming the multisubunit Rh structures in the RBC membrane.

The authors are grateful to Dr A. Blancher for his monoclonal antibody LOR-15C9 against RhD and to Dr A.E.G.Kr. von dem Borne for his monoclonal antibody 2D10 against Rh50. We thank G. Halverson for immunoblot analysis and T. Huima-Byron, Y. Oskov, and R. Ratner for photoprints and computer graphics.