Nt1230 Unit 3 Assignment 1

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Edexcel BTEC - Assignment Cover Sheet

Qualification
Unit number(s) and title(s)
BTEC
Unit 11 Systems Analysis and Design
Learner name Assessor name

Geraldine Hudson
Date issued
Deadline
Submitted on
W/C 19/01/15
W/C 23/02/15
(Thursday 4pm)

Assignment title

What is Systems Analysis?
In this assessment you will have opportunities to provide evidence against the following criteria.
Indicate the page numbers where the evidence can be found.

Criteria reference
To achieve the criteria the evidence must show that the learner is able to:

Task no.

Evidence
P4
Carry out a structured analysis of a specified business process

1

P5
Produce a requirements specification for a business process

1

M2
Suggest alternative solutions

1

D1…show more content…

Scenario
Micronics is a computer games company that designs and produces multi -platform computer games. It sells directly into the retail, Argos industry (GAME, WH Smith) and on-line retailers such as Amazon and GameStop.co.uk. There is a team of three sales people who report into one sales manager. The sales team have the geographical remit of the UK. This sales team is assisted by three sales administration assistants, also managed by the Sales Manager, who log the sales team’s orders and send them to production. The sales administrators do not get involved with selling new products – only with providing administrative support to the Sales team.

The sales administration team is quite formally structured, with each Sales Administrator responsible for inputting a particular sales persons orders.
The Managing Director of Micronics feels that there are some issues with the service that the Sales Administrators offer to the Salesmen and wants to improve it. He did employ an external consultant to look at this, but due to budget restrictions, has recently had to withdraw.
You have been asked to take over the investigation and complete the analysis and design. You have limited experience with this and therefore have to investigate and document the main principles of early systems analysis. You have been given some notes and transcripts of interviews (please see end of this assignment brief).

Tasks

Scenario
You have recently submitted an investigation

The Human Rh50 Glycoprotein Gene

STRUCTURAL ORGANIZATION AND ASSOCIATED SPLICING DEFECT RESULTING IN Rhnull DISEASE*

  1. Cheng-Han Huang‡
  1. From the Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021

Abstract

The Rh (Rhesus) protein family comprises Rh50 glycoprotein and Rh30 polypeptides, which form a complex essential for Rh antigen expression and erythrocyte membrane integrity. This article describes the structural organization of Rh50 gene and identification of its associated splicing defect causing Rhnulldisease. The Rh50 gene, which maps at chromosome 6p11–21.1, has an exon/intron structure nearly identical to Rh30 genes, which map at 1p34–36. Of the 10 exons assigned, conservation of size and sequence is confined mainly to the region from exons 2 to 9, suggesting thatRH50 and RH30 were formed as two separate genetic loci from a common ancestor via a transchromosomal insertion event. The available information on the structure of RH50facilitated search for candidate mutations underlying the Rh deficiency syndrome, an autosomal recessive disorder characterized by mild to moderate chronic hemolytic anemia and spherostomatocytosis. In one patient with the Rhnull disease of regulator type, a shortened Rh50 transcript lacking the sequence of exon 7 was detected, while no abnormality was found in transcripts encoding Rh30 polypeptides and Rh-related CD47 glycoprotein. Amplification and sequencing of the genomic region spanning exon 7 revealed a G → A transition in the invariant GT motif of the donor splice site in both Rh50 alleles. This splicing mutation caused not only a total skipping of exon 7 but also a frameshift and premature chain termination. Thus, the deduced translation product contained 351 instead of 409 amino acids, with an entirely different C-terminal sequence following Thr315. These results identify the donor splicing defect, for the first time, as a loss-of-function mutation at theRH50 locus and pinpoint the importance of the C-terminal region of Rh50 in Rh complex formation via protein-protein interactions.

The Rh (Rhesus) protein family is currently known to consist of three erythroid-specific integral membrane proteins, the Rh50 glycoprotein and two Rh30 (RhD and RhCE) polypeptides (1-4). Although their genetic loci are mapped on chromosomes 6p11–21.1 and 1p34–36, respectively, Rh50 and Rh30 share a clear sequence homology (36% overall identity) and a similar 12-transmembrane (TM)1 topology (50% identity in the putative α-helices) (5-8). As nonglycosylated and palmitoylated proteins, RhD and RhCE each contain 417 amino acids, serving as the carriers of D and CcEe blood group antigens (5-7). By contrast, the 409-amino acid Rh50 glycoprotein in itself does not carry Rh antigens but rather interacts with Rh30 polypeptides to form a protein complex, thereby functioning as a coexpressor to facilitate Rh antigen disposition in the erythrocyte membrane (8-10).

Apart from being a structural unit of Rh antigen expression, the Rh50 and Rh30 proteins appear to possess some hitherto undefined roles essential for the function and integrity of plasma membranes. This proposal is highlighted primarily by the occurrence of Rh deficiency syndrome, a rare autosomal recessive disorder characterized by a chronic hemolytic anemia of varying severity, a hereditary spherostomatocytosis, and multiple membrane abnormalities (1-3). The Rh deficiency syndrome exists in two conditions in which a complete absence of all Rh antigens defines the Rhnull status and a barely detectable presence defines the Rhmod phenotype (11,12). Both conditions exhibit an absence or weakened expression of several other membrane glycoproteins or associated antigens, including Rh50, CD47, LW, Duffy (Fy5), and glycophorin B (GPB for SsU) (1-3). Therefore, the Rh deficiency syndrome can be regarded as a disorder of impaired protein-protein interactions.

As shown by family studies, Rh deficiency is almost invariably associated with consanguinity and can occur on different genetic backgrounds (11, 12). The amorph type of Rhnull is thought to arise by silencing mutations at the RH30 locus encoding RhD and RhCE polypeptides, but its underlying molecular defect has remained to be determined (13-15). In contrast, the regulator Rhnull and Rhmod phenotypes are considered to result from suppressor or “modifier” mutations independent of theRH30 locus (16). The genuine interaction of Rh50 with Rh30 proteins in Rh complex formation points to RH50 locus as a primary candidate responsible for the suppressor forms of Rh deficiency. To facilitate the identification of such suppressor mutations, the organization of Rh50 gene has now been delineated. Here, I describe the exon/intron structure of the Rh50 gene and identification of its associated splicing defect as a loss-of-function mutation in one Rhnull patient. The findings reported herein correlate the disease phenotype with an impaired Rh complex formation and provide evidence for the importance of the C-terminal region of Rh50 participating in protein-protein interactions.

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EXPERIMENTAL PROCEDURES

Blood Samples

Blood samples from normal human blood donors with RhD-positive (RhD+) and RhD-negative (RhD) phenotypes (defined by DCe/DCe anddce/dce genotypes) were used as controls. The Rhnull blood sample was obtained from a Japanese patient (T. T.). Preliminary studies showed that the propositus was a homozygote for the regulator type of Rhnull disease and no Rh antigen was detectable by serologic testing. Furthermore, Southern blot analysis demonstrated that the RH30 locus was grossly intact without apparent gene deletion or rearrangement (15).

Nucleic Acid Isolation and Southern Analysis

Total RNA was isolated from reticulocyte polysomes using the differential cell lysis method (17), followed by extraction with the Trizol reagent (Life Technologies, Inc.). Genomic DNA was prepared from leukocyte pellets, as described previously (18). Southern blot analysis was performed using Rh50, Rh30, and CD47 cDNA probes generated with gene-specific primers (see below) and labeled with [α-32P]dCTP (NEN Life Science Products).

Characterization of Exon/Intron Structure of the Rh50 Gene

To determine the structural organization of the Rh50 gene, genomic DNA from a normal person was digested separately with restriction endonucleases EcoRV, HincII,PvuII, SmaI, SspI, andStuI. The total digests of each restriction enzyme were ligated to the same adaptor to generate a genomic library using the Marathon amplification kit (CLONTECH). The exon and its adjacent intron sequences were then amplified in two steps using the Taq DNA polymerase chain reaction (PCR) (19). The first step employed the adaptor primer (AP1) and a Rh50 gene-specific primer (GSP1), whereas in the second step, nested AP2 and GSP2 were used. The resultant products were analyzed by agarose gel and sequenced after purification by 5% polyacrylamide gel electrophoresis. When new sequence information became available, new primers were designed for further bidirectional walking (Table I).

Table I

synthetic oligonucleotides for analysis of Rh50 glycoprotein gene

RT-PCR Analysis of Rh50, Rh30, and CD47 Transcripts

To determine the structure and expression of Rh50, Rh30 and CD47 transcripts in normal and Rhnull erythroid cells, cDNAs were synthesized from total RNA and amplified by RT-PCR, as described (20). The cDNA was reverse-transcribed with an oligo(dT) primer or a gene-specific primer located in the 3′-untranslated region (3′-UTR); the entire coding sequence was then amplified in two overlapping segments with four 5′ amplimers. All nucleotide (nt) positions of sense (s) and antisense (a) primers are counted from the first base of ATG codon in the respective cDNAs (5-8, 21). The Rh50 primers were: 1) 3′-UTR, 5′-AATGGGAAAGGAAGCTGGAGAGCA-3′ (nt 1321–1298); 2) amplimers: 1s, 5′-AGTGTGCCTCTGTCCTTTGCCACA-3′ (nt −27 to −4, 5′-UTR of exon 1); 5a, 5′-CTGTTTGTCTCCAGGTTCAGCAAT-3′ (nt 708–685, exon 5); 4s, 5′-GAAGAGTCCGCATACTACTCAGAC-3′ (nt 601–624, exon 4); 7s, 5′-CCACTTTTTACTACTAAACTGAGG (nt 946–969, exon 7); and 10a, 5′-CCATGTCCATGGAACTGATTGTCA-3′ (nt 1256–1233, exon 10). The Rh30 primers were: 1) 3′-UTR of RhD, 5′-GTATTCTACAGTGCATAATAAATGGTG-3′ (nt 1458–1432, exon 10); and 3′-UTR of RhCE, 5′-CTGTCTCTGACCTTGTTTCATTATAC-3′ (nt 1388–1363, exon 10); 2) amplimers: 1s, 5′-ATGAGCTCTAAGTACCCGCGGTCTG-3′ (nt 1–25, exon 1); 5a, 5′-TGGCCAGAACATCCACAAGAAGAG-3′ (nt 663–640, exon 5); 4s, 5′-CCAAAATAGGCTGCGAACACGTAGA-3′ (nt 515–539, exon 4), and 10a, 5′-TTAAAATCCAACAGCCAAATGAGGAAA-3′ (nt 1254–1228, exon 10). The CD47 primers were: 1) 3′-UTR, 5′-TCACGTAAGGGTCTCATAGGTGAC-3′ (nt 1120–1197); 2) amplimers: Is, 5′-ATGTGGCCCCTGGTAGCGGCGCT-3′ (nt 1–23); Ia, 5′-CACTAGTCCAGCAACAAGTAAAGC-3′ (nt 555–534); IIs, 5′-CTCCTGTTCTGGGGACAGTTTGGT-3′ (nt 460–483); and IIa, 5′-CAAATCGGAGTCCATCACTTCACT-3′ (nt 1001–977).

Amplification and Analysis of the Genomic Region Encompassing Exon 7 of Rh50 Gene

To assay the donor splice site mutation, the genomic region spanning exon 7 of RH50 in normal and Rhnull was amplified. For PmlI digestion, the fragment was amplified with intron primers 6s and 7a: intron 6s, 5′-GCCCAGCTATAGCTGTGTTTCAGT-3′ (nt −80 to −56 upstream of exon 7); and intron 7a, 5′-CTAATGATCTTCTCTCAGGCGCGT-3′ (nt 128–152 downstream of exon 7). For restriction analysis with NlaIII, the fragment was amplified with exon 7 primer 7s (nt 946–969, see above) and intron 7 primer 7a′, 5′-ATGGGACCACAGGGGCTGA-3′ (nt 22–40 downstream of exon 7).

Direct Nucleotide Sequencing and Sequence Analysis

All amplified cDNA and genomic DNA products were purified by native 5% polyacrylamide gel electrophoresis and sequenced with either amplimers or nested primers. Nucleotide sequence determination was carried out using fluorescent dye-tagged chain terminators on an automated DNA sequencer (model 373A, Applied Biosystems). The resultant nucleotide sequences were analyzed by the DNASIS program (Hitachi), and the deduced amino acid sequences were assessed for hydropathy character using the Kyte-Doolittle plotting method (22).

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RESULTS

Organization of Rh50 Gene and Comparison with Rh30 Gene

To delineate the structural organization of the Rh50 gene, a bidirectional walking approach was taken to retrieve unknown sequences (Fig.1A). 40 synthetic primers that cover various coding sequences (Table I) were used in combination to amplify the adaptor-ligated, restriction enzyme-specific genomic libraries. Fig. 1A shows a representative panel of the resultant Rh50gene products, each spanning a unique exon/intron junction. They range in size from several hundred base pairs (bp) to several kilobase pairs, depending on the distribution of restriction sites. Sequencing of these amplified products revealed the features of the Rh50 gene and confirmed no coamplification from the related Rh30 genes.

Figure 1

Amplification of exon/intron junctions and organization of the Rh50 gene. A, strategy for amplification of exon/intron junction segments. The transcript and genomic structures of Rh50 are schematically shown (not to scale). Initiation ATG and stop TAA codons in the cDNA and upstream ATG leading a potential open reading frame in the genomic DNA are denoted. Bent arrowsillustrate the 40 primers anchored to 10 exons in either sense (s) or antisense (a) direction (Table I). Shown below is a representative 1.8% agarose gel electrophoresis of sequenced genomic products. The exon (E)/intron (IVS) content and restriction enzyme usage of amplified fragments are indicated. Note that lanes 8 and 13are products amplified directly from total genomic DNA, which encompass the whole intron 4 and intron 6, respectively. Bands seen in other lanes each were obtained by two rounds of PCR using AP1+GSP1 and AP2+GSP2 (Table I). M, λ (HindIII) and φX174 (HaeIII) DNA markers. B, organization of the Rh50 gene and comparison with the Rh30 gene. Exons are denoted bysolid or open bars and introns by broken lines (not to scale). The size of coding sequence for each exon (in base pairs) is shown; exon 1 is counted from ATG and exon 10 ends before the TAA codon (marked by asterisks).

The translated sequence of Rh50 was found to be distributed in 10 exons whose size ranges from 15 (exon 10) to 184 bp (exon 2) (Fig.1B). This global organization is strikingly similar to that of the Rh30 genes (23, 24) and is essentially conserved in the Rh50 homologues from the mouse and Caenorhabditis elegans.2Comparison of Rh50 with Rh30 showed that their sequence homology is confined mainly to exons 2–9, whereas their 5′ or 3′ regions share little or no sequence similarity. The size of all internal exons except exons 7 and 8 was conserved, and exon 2 of Rh50 was missing codon AGT for Ser99, which is present in Rh30 genes (5-7). Thus, Rh50 and Rh30 show the same assignment of exon/intron junctions except for a difference in their exon 7/exon 8 boundaries (Fig.1B). The 5′ region of Rh50 has several putative cis-acting elements (Fig. 2), including the TATA boxes that are absent from the proximal promoter of both RhD and RhCE (23, 24). Multiple transcription initiation sites were identified between the two Ets elements. This mapping result was consistent with the assignment of ATG initiation codon noted in bone marrow Rh50 mRNAs (8), although the genomic sequence indicated a potential occurrence of another in-frame ATG codon upstream (nt −96 to −94) (Fig. 2). A detailed study of the Rh50 gene, including the mapping of its introns and dissection of its promoter activity and transcription initiation sites, will be described elsewhere.3

Figure 2

Nucleotide sequence of the 5′ portion of the Rh50 gene. The 5′ region and exon sequences are shown byuppercase letters and the intron 1 sequence bylowercase letters. For brevity, the intron 1 sequence, whose size is grater than 15 kilobase pairs in size, is omitted (shown bydots). Putative cis-acting motifs in the promoter are marked and underlined. Note that TATA boxes are not found in the promoter of both RhD and RhCE genes (23, 24). Note also that there is a strong strand asymmetry in the region. Multiple transcription initiation sites occur between the two putative Ets binding sites (see Footnote 3). The first position of ATG codon assigned for translation initiation of the erythroid-specific Rh50 protein (8) is denoted. The encoded amino acids of exon 1 and exon 2 (partial) are shown below the nucleotide sequence.

Sequence of Splice Sites and Exon/Intron Junctions in the Rh50 Gene

Fig. 3 schematically shows the nucleotide sequence of splice sites as well as the structure of exon/intron junctions in the Rh50 gene. All the 5′ donor and 3′ acceptor splice sites conform to the “GT-AG” rule and possess the consensus splicing signals (25). Of the 10 exons identified, only exon 6 is symmetrical, having intraexon codons GTT (Val270) and ACT (Thr315) at its 5′ and 3′ ends, respectively, whereas the other exons have either one or two split interexon codons (Fig. 3). One potential consequence of this type of exon/intron arrangement is that skipping of any single internal exon, except exon 6, during the splicing of Rh50 pre-mRNA would result in a shift in open reading frame and, therefore, alter the encoded amino acid sequence downstream of the skipped exon.

Figure 3

Sequence of exon/intron junctions and assignment of splice sites in the Rh50 gene. The 409-amino acid coding sequence of Rh50 gene is distributed in 10 exons (boxed). The nucleotide positions marking the beginning and end of each exon are numbered: nt 1 denotes the first nucleotide of erythroid ATG initiation codon and nt 1230 the third base of TAA stop codon. “aataaa” indicates one of the polyadenylation signals present in the 3′-UTR. Exon sequences are denoted byuppercase letters, whereas intron sequences, including the 3′-acceptor and 5′-donor splice sites, are indicated by lowercase letters. Interval exon sequences are omitted (shown bydots). Amino acids encoded by the respective exon/exon boundaries are indicated below the middle position of the triplet code.

Expression of Rh50, Rh30, and CD47 mRNAs in Normal and Rhnull Cells

To identify the molecular defect underlying the Rhnull disease, the expression of candidate genes encoding the Rh50, Rh30, and CD47 proteins was characterized by RT-PCR and nucleotide sequencing. The full-length cDNA of Rh30 or CD47 was readily detectable in normal and Rhnull erythroid cells (gels not shown), indicating a comparable expression of the corresponding mRNA. Sequencing showed that the Rh30 or CD47 cDNA from Rhnull was normal and that the Rh30 cDNA contained both RhD and RhCe, indicating that the patient was aDCe/DCe homozygote. Definition of this Rh genotype by transcript analysis was in full agreement with the result of DNA typing by SphI polymorphisms (15). These data showed that theRH30 or CD47 locus itself is not responsible for the disease phenotype.

However, RT-PCR analysis of Rh50 gene expression in erythroid cells revealed an important difference between the normal and Rhnull patient. Although there was no apparent change in size of the 5′ portion of Rh50 cDNA encompassing exons 1–5, the 3′ portion of Rh50 cDNA encompassing exons 4–10 always showed a truncation in the Rhnull patient (Fig.4A). This finding indicated that the Rh50 mRNA from Rhnull could be an aberrantly spliced form lacking a portion of the 3′ sequence. Indeed, sequencing showed that the 122-bp sequence of exon 7 was excluded from the truncated cDNA, resulting in the connection of exon 6 to exon 8 (Fig. 4B). To determine whether the skipping was complete or partial, a 3′ RACE reaction was carried out using 7 s and 3′-UTR primers. A cDNA product of expected size (376 bp) was found in normal controls but not in the Rhnull patient (Fig.4C), indicating that no splicing of exon 7 occurred for the Rh50 primary transcript. Further studies showed that this exon skipping was not seen in 15 normal subjects nor in other Rhnullpatients examined; thus, it could not be a constitutive splicing or regulated alternative splicing event.

Figure 4

Analysis of Rh50 transcript expression in normal and Rhnull erythroid cells. RT-PCR analysis of Rh50 transcript was carried out using 3′-UTR primer for cDNA synthesis and two pairs of amplimers for cDNA amplification. The location, direction, and designation of primers with respect to the structure of Rh50 are specified. A, agarose gel electrophoresis of amplified Rh50 cDNA products from RhD+, RhD, and Rhnull. The size of segment 4s-10a from Rhnull is smaller than that of controls, indicating a deletion in the region spanning exons 5–10. Note that the Rhnull lanes were overloaded. B, nucleotide profiles of the exon/exon boundary associated with exon skipping. Exon boundary is indicated by a vertical arrow. In normal, exon 6 is joined to exon 7, whereas in Rhnull exon 7 is absent, resulting in exon 6 to exon 8 connection. C, 3′ RACE assay for the functional splicing of exon 7 in Rh50 pre-mRNAs. A primer anchored in exon 7, 7s, was coupled with 3′-UTR primer for 3′ RACE reaction. The expected cDNA product of 376 bp is clearly seen in control lanes but not the Rhnull lane, confirming a complete exclusion of exon 7 from the latter.

Identification of Rhnull-associated Donor Splice Site Mutation in Rh50 Gene

The complete absence of exon 7 associated with Rh50 cDNA suggested strongly that either a splicing defect or a genomic deletion was present in the cognate gene. To define the nature of the underlying mutation, amplification from Rhnull genomic DNA of a segment encompassing exon 7 of the Rh50 gene was attempted. A fragment of 354 bp in size was detected, excluding the possibility of gene deletion. Sequencing of this fragment on both strands led to the identification of a single G → A mutation in the invariant GT element (+1 position) of the 5′ donor splice site attached to exon 7 (Fig. 5A). Sequencing of other exon/intron junctions amplified with intron-specific primers (data not shown) confirmed this mutation to be the only structural alteration in the Rh50 gene.

Figure 5

Identification of Rhnull-associated donor splice site mutation in intron 7 of Rh50 gene. A, amplification and sequencing of the genomic segments encompassing exon 7 of the Rh50 gene. The G → A transition at the +1 position of intron 7 in the nucleotide profiles is indicated by two vertical arrows. Shown at bottom are thePmlI site located exactly at exon 7/intron 7 junction (CAC↓GTG) of wild-type Rh50 and the NlaIII site (↓CATG) in mutant Rh50, resulting from the 5′ donor splice site mutation. The direction and position of primers are indicated. B, a diagnostic analysis of the donor splice site mutation withPmlI and NlaIII enzymes. The 6s-7a fragment was cleavable with PmlI in normal but not Rhnull. In contrast, the 7s-7a′ fragment of Rhnull has an extraNlaIII site. C, Southern blot analysis of native genomic DNAs from normal person and Rhnull patient. Genomic DNAs were digested the enzymes indicated and hybridized with an exon7/intron 7 junction probe. The PmlI cleavable fragments are seen in normal persons but not in Rhnull patient.

Because the mutation abolished a PmlI restriction site (CAC↓GTG) (Fig. 3) and introduced a novel NlaIII site (↓CATG), a direct diagnostic assay was performed on amplified exon 7-containing fragments. The two enzymes showed an opposite cleavage pattern in normal and Rhnull fragments (Fig.5B), confirming the mutation at the splicing junction. To demonstrate that loss of the PmlI site was not caused by PCR spurious mutations, Southern blot of native genomic DNAs was hybridized with a probe spanning the exon7/intron 7 junction. As shown, thePmlI specific band was seen in normal but not in Rhnull (Fig. 5C). Given the observation of no dosage reduction in RH50, these results confirmed that the patient is homozygous for the G → A splicing mutation. Such a genotype assignment is consistent with the inheritance of Rhnull syndrome in an autosomal recessive fashion.

Deduced Primary Sequence and Predicted Membrane Topology of Rh50 Mutant Protein

To gain information on the primary structure of Rh50 glycoprotein, the Rhnull-associated Rh50 cDNAs were sequenced to completion. Compared with normal Rh50, no point mutation other than an absence of the sequence encoded by exon 7 was observed in the Rhnull patient (Fig.6A). Because exon 7 is asymmetric in codon distribution at the 5′ side (Fig. 3), its complete skipping and the subsequent joining of exon 6 with exon 8 inevitably resulted in an open reading frame shifting (Fig. 6A). In turn, the deduced translation product would be truncated and prematurely terminated, containing only 351 amino acid residues. This includes the loss of 41 amino acid residues encoded in exon 7 and gain of an entirely new sequence of 36 residues following Thr315(Fig. 6A).

Figure 6

Amino acid sequence and predicted membrane topology of mutant Rh50 in Rhnull disease. A, comparison of the primary structure between the wild-type (wt) and mutant (mt) Rh50 glycoproteins. The mutant lacks 41 amino acids (dashes) encoded by exon 7, but gains a new 36-amino acid sequence (bold) due to a frameshift and premature termination (preterm). Note that in both the mutant and wild types, the amino acid at position 242 is occupied by Asn (N with an asterisk) but not by Asp (D) as reported (8). This Asn is seen in all unrelated normal and Rhnull individuals examined (n> 15). B, model for membrane topology of the mutant Rh50 protein. Also shown is the hydropathy profile of wild-type Rh50 with 12 TM domains connected by short loops on either side of the lipid bilayer. “Y” indicates the N-glycan on Asn37 (9). The mutant Rh50 protein lacks the last two TM domains and carries an extended C-terminal sequence most likely facing the cytoplasmic space. Its features, including the underlying defect and associated structural alterations, are summarized at right margin.

Compared with the wild-type Rh50 protein (8), hydropathy plot analysis of the mutant form suggested two possible alterations in membrane organization of the C-terminal region (Fig. 6B). (i) Deletion of the exon 7-coding sequence abolishes the 5th intracellular loop as well as the 11th TM segment. (ii) The inherent frameshift and premature termination further eliminates the last TM domain, and the resulting new sequence would face the cytoplasmic side due to lack of a continuous stretch of hydrophobic residues. Apparently, loss of a normal C-terminal portion of the Rh50 protein is the major cause for the perturbation of Rh complex formation in the Rhnullerythrocyte.

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DISCUSSION

Rh50 glycoprotein is a critical coexpressor of Rh30 polypeptides, the carriers of erythrocyte Rh antigens (1-4). Here, the exon/intron structure of Rh50 gene has been delineated, which should facilitate identification of mutations underlying the suppressor forms of Rh deficiency syndrome. A homology-based approach coupling with bidirectional walking revealed that Rh50 is a single copy gene with 10 exons and has a global organization strikingly similar to its related Rh30 members (23, 24). Both the structural conservation and sequence homology of the two genes are confined mainly to exons 2–9, while their 5′ and 3′ regions, including the promoter and untranslated sequences, share little or no similarity. Since Rh50 and Rh30 genes are located on different chromosomes (5-8), these findings suggest that the two genetic loci might be formed by a rare transchromosomal insertion event. Our recent studies suggest that Rh50 and Rh30 genes originated from a common ancestor and were linked to each other following their initial duplication; later, one was translocated and diverged as the independent locus on a separate chromosome.4 Comparative analysis of the Rh50 and Rh30 gene orthologues in lower organisms should help decipher the evolutionary pathway ultimately leading to the establishment of two genetic loci encoding the Rh family proteins inHomo sapiens.

The extreme rareness, recessive nature, and consanguineous background of Rh deficiency syndrome (11, 12) point to a heterogeneous spectrum of the underlying mechanisms. At present, the molecular defect atRH30 locus responsible for the amorph type of Rhnull remains unknown (13-15). Nevertheless, several lines of evidence suggest that the RH50 locus is the prime target of suppressor mutations resulting in the regulator Rhnull disease. (i) Rh50 is thought to directly interact with Rh30, and the deficiency of the two proteins in the plasma membrane occurs in parallel (9, 26). (ii) Despite a close link of Rhnull with absence or deficiency in GPB, Duffy, or LW, the erythrocytes lacking these glycoproteins per se exhibit no change in the Rh antigen expression and no apparent perturbations in membrane physiology and cell morphology (27-30). Presumably these proteins are casually associated components not essential for the interaction and membrane assembly of Rh family proteins. (iii) Although CD47 is also reduced in Rhnull state, its low level of expression is restricted to erythroid cells but not to other hematopoietic cells (31, 32), suggesting that CD47 deficiency occurs as the consequence of, rather than the cause for, the defect in Rh complex formation. (iv) More recently, two small DNA deletions causing frameshift in the Rh50 gene have been found to be associated with the regulator Rhnull phenotype in unrelated patients (16).

Our previous studies showed that this Rhnull patient had a grossly intact RH30 locus occurring in the form ofDCe/DCe haplotype combination (15). The present study confirmed this assignment and showed further that the RH30locus gave rise to expression of both RhD and RhCe transcripts with sequences identical to that from normal subjects. These results, together with the identification of a normal CD47 gene, exclude the involvement of mutations of RH30 or CD47 locus in this Rhnull patient. However, transcript analysis showed that there was no expression in the Rhnull cells of any full-length form of Rh50 mRNAs except the shortened one specifically lacking the sequence of exon 7. Genomic sequencing revealed the occurrence of a homozygous G → A mutation in the invariant GT element of 5′ donor splice site as the only alteration in the Rh50 gene. These findings establish the pre-mRNA splicing defect, for the first time, as the suppressor mutation ofRH50 leading to a loss-of-function phenotype characteristic of the regulator form of Rhnull disease.

Mutations in the GT and AG motifs of the donor and acceptor splice sites, the cis-acting elements essential for pre-mRNA splicing (33), portray an important mechanism for the origin of human genetic diseases (34). The donor splice site mutation described here has caused a complete skipping of exon 7 from the mature form of Rh50 mRNA in the Rhnull patient. Significantly, such a splicing event not only excluded a coding sequence for 41 amino acids but resulted in a frameshift after the codon for Thr315 and a premature chain termination after the codon for Ile351. Therefore, the deduced Rh50 mutant protein contains only 351 amino acids, including a stretch of 36 new residues at the C terminus. Correlation of these primary changes with regulator Rhnulldisease provides new insight regarding how different mutations might act as suppressors to disrupt or modify the protein-protein interactions that dictate the Rh complex formation.

Prior studies suggested that there may be a direct contact between Rh50 and Rh30 via their N-terminal sequences (9, 10). Nevertheless, additional interacting sites are likely to be present in the Rh protein complex. For the Rh50 mutant reported here, its only difference from the wild-type lies C-terminal to the 10th putative TM domain (Fig. 6). This suggests that the C-terminal half may also participate in the interaction directly and/or confer required conformation to stabilize that interaction. In support of this notion, we have identified in unrelated Rhnull patients several missense mutations that are clustered in the C-terminal region of the Rh50 protein.4 It is of further interest to note that such mutations all target the TM domains in the C-terminal half that are conserved in the Rh50 homologues from the mouse to C. elegans. Currently, little is known about how the disruption of the Rh protein complex causes the multiple facets of structural and functional abnormalities in the Rh-deficient erythrocytes. There is also a lack of general information regarding the involvement and coordination of possible intracellular factor(s) in the functioning of the Rh membrane complex. A full description of Rhnulldisease mutations and assessment of their phenotypic effects in model systems, such as C. elegans, should lead to a better understanding of the membrane assembly and structure/function relations of the Rh family of proteins.

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ACKNOWLEDGEMENT

I am particularly grateful to Y. Okubo and M. Reid for providing and typing the Rhnull blood sample used in this investigation. I thank Y. Chen for technical assistance, and T. Ye for help in the construction of human Marathon genomic libraries. I also thank O. O. Blumenfeld and C. Redman for comments on the manuscript.

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Footnotes

  • ↵* This work was supported in part by National Institutes of Health Grant HL54459.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF031548, AF031549, AF031550, and AF031551.

  • ↵‡ To whom correspondence should be addressed: Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E. 67th St., New York, NY 10021. Tel.: 212-570-3388; Fax: 212-737-4935; E-mail: chuang{at}nybc.org.

  • ↵1 The abbreviations used are: TM, transmembrane; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; UTR, untranslated region; nt, nucleotide(s); bp, base pair(s); RACE, rapid amplification of cDNA ends.

  • ↵2 Z. Liu and C.-H. Huang, unpublished observations.

  • ↵3 Z. Liu and C.-H. Huang, manuscript in preparation.

  • ↵4 C.-H. Huang, J. Cheng, Y. Chen, and Z. Liu, unpublished observations.

  • Received September 23, 1997.
  • Revision received October 27, 1997.
  • The American Society for Biochemistry and Molecular Biology, Inc.

REFERENCES

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