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Biomedical subjects

E Smart

Publications and source records attributed to E Smart.

At least 19 recordsLinked to original sources
PubMed2005

The Abantu phenotype in the ABO blood group system is due to a splice-site mutation in a hybrid between a new O1-like allelic lineage and the A2 allele.

BACKGROUND AND OBJECTIVES: Many phenotypic variations in the expression of blood group A have been explained by variations in gene structure, but unresolved samples are frequently encountered in the reference laboratory. Among ABO subgroups, A(bantu) has the highest frequency in a specified population. The molecular basis of this phenotype is now described. MATERIALS AND METHODS: Blood from Black donors phenotyped as A(bantu) was subjected to genomic ABO screening and direct sequencing of polymerase chain reaction (PCR)-amplified ABO exons 1-7 and introns 2-6. Total RNA was extracted and ABO cDNA was synthesized by reverse transcription (RT)-PCR. Control material comprised Black South African, Swedish, Jordanian and Brazilian blood samples with common phenotypes. RESULTS: Genomic ABO typing indicated the presence of an A(2) allele in each A(bantu) donor, in combination with an O allele. No previously reported mutations associated with weak A or B expression were found. Direct sequencing indicated the common A(2) sequence with a single nucleotide deletion (AGGT>AGT) at the exon 4/intron 4 junction, predicted either to disrupt the reading frame (resulting in a premature stop codon) or to cause erroneous splicing (resulting in the exclusion of exon 4 from the mRNA). O mRNA, but no transcripts from the A(bantu) allele, could be detected. Surprisingly, the splice-site mutation was also found in approximately 5% of O alleles in Black South Africans, but not in other blood donors, or in non-O(1) alleles. Utilizing intron polymorphisms, the A(bantu) allele was shown to be a recombination between a new allelic lineage (O(1bantu)) and A(2), with a cross-over region near exon 5. CONCLUSION: The A(bantu) phenotype is caused by an O(1bantu)-A(2) hybrid at the ABO locus.

ABO Blood-Group System↗
PubMed2000

The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype.

Antigens of the Rh blood group system are encoded by 2 homologous genes, RHD and RHCE, that produce 2 red cell membrane proteins. The D-negative phenotype is considered to result, almost invariably, from homozygosity for a complete deletion of RHD. The basis of all PCR tests for predicting fetal D phenotype from DNA obtained from amniocytes or maternal plasma is detection of the presence of RHD. These tests are used in order to ascertain the risk of hemolytic disease of the newborn. We have identified an RHD pseudogene (RHD psi) in Rh D-negative Africans. RHDpsi contains a 37 base pair (bp) insert in exon 4, which may introduce a stop codon at position 210. The insert is a sequence duplication across the boundary of intron 3 and exon 4. RHDpsi contains another stop codon in exon 6. The frequency of RHDpsi in black South Africans is approximately 0.0714. Of 82 D-negative black Africans, 66% had RHDpsi, 15% had the RHD-CE-D hybrid gene associated with the VS+ V- phenotype, and only 18% completely lacked RHD. RHDpsi is present in about 24% of D-negative African Americans and 17% of D-negative South Africans of mixed race. No RHD transcript could be detected in D-negative individuals with RHDpsi, probably as a result of nonsense-mediated mRNA decay. Existing PCR-based methods for predicting D phenotype from DNA are not suitable for testing Africans or any population containing a substantial proportion of people with African ethnicity. Consequently, we have developed a new test that detects the 37 bp insert in exon 4 of RHDpsi. (Blood. 2000; 95:12-18)

Amino Acid Sequence↗
PubMed2000

An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype.

Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.

3' Untranslated Regions↗
PubMed1999

Production and characterization of anti-kell monoclonal antibodies using transfected cells as the immunogen.

Monoclonal antibodies (Mabs) to blood group antigens are valuable as diagnostic reagents for typing red blood cells (RBCs) in the clinical setting, and for structure-function studies of proteins. Here, we report a powerful system that enabled us to produce Mabs to blood group antigens. A murine erythroleukaemia (MEL) cell line expressing Kell protein, a transmembrane glycoprotein that carries a number of clinically relevant antigens, was used as a novel immunogen. Mabs with different specificities to the Kell protein were produced from a single mouse fusion: an anti-Jsb (MIMA-8), and two antibodies (MIMA-9 and MIMA-10) with novel specificities, that reacted with RBCs with the common Kell phenotype but not with RBCs with K+k- or Kp(a+b-) or K0 phenotypes. The non-reactivity with both K+k- or Kp(a+b-) RBCs implied that the epitope was influenced by the molecular changes associated with an absence of the k or Kpb antigens. MIMA-8 is the first example of a Mab anti-Jsb and was used in the clinical laboratory for screening donor RBCs for Js(b-) blood and for typing RBCs from patients even when the RBCs were coated with anti-IgG as is the case in autoimmune haemolytic anaemia. Heavy and light chain variable regions of MIMA-8 were cloned and the sequence is given. This study illustrates the potential of this novel immunization approach for making monoclonal antibodies to blood group antigens.

Animals↗
PubMed1998

The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis.

BACKGROUND: VS and V are common red cell antigens in persons of African origin. The molecular background of these Rh system antigens is poorly understood. STUDY DESIGN AND METHODS: Red cells from 100 black South Africans and 43 black persons from Amsterdam, the Netherlands, were typed serologically for various Rh system antigens. Allele-specific polymerase chain reaction and sequencing of polymerase chain reaction products were used to analyze C733G (Leu245Val) and G1006T (Gly336Cys) polymorphisms in exons 5 and 7 of RHCE and the presence of a D-CE hybrid exon 3. RESULTS: The respective frequencies of all VS+ and of VS+ V-(r's) phenotypes were 43 percent and 9 percent in the South Africans and 49 percent and 12 percent in the Dutch donors. All VS+ donors had G733 (Val245), but six with G733 were VS- (4 V+w, 2 V-). The four VS- V+w donors with G733 appeared to have a CE-D hybrid exon 5. T1006 (Cys336) was present in 12 percent and 16 percent of donors from the two populations. With only a few exceptions, T1006, a D-CE hybrid exon 3, and a C410T (Ala137Val) substitution were associated with a VS+ V-phenotype ((C)ces or r's haplotype). Two VS+ V-individuals, with the probable genotype, (C)ces/(C)ces), were homozygous for G733 and for T1006. CONCLUSIONS: It is likely that anti-VS and anti-V recognize the conformational changes created by Val245, but that anti-V is sensitive to additional conformational changes created by Cys336.

Alleles↗
PubMed1997

Missense mutation of FUT1 and deletion of FUT2 are responsible for Indian Bombay phenotype of ABO blood group system.

The Bombay phenotype fails to express the ABH antigens of ABO blood group system on red blood cells and in secretions because of a lack in activities of the H gene (FUT1)- and Secretor gene (FUT2)-encoded alpha (1,2)fucosyltransferases. In this study, we have examined the FUT1 and the FUT2 from three unrelated Indian individuals with the Bombay phenotype. These three individuals were found to be homozygous for a T725G mutation in the coding region of the FUT1, which inactivated the enzyme activity. In addition, we did not detect any hybridized band corresponding to the FUT2 by Southern blot analysis using the catalytic domain of the FUT2 as a probe, indicating that the three individuals were homozygous for a gene deletion in the FUT2. These results suggest that the T725G mutation of FUT1 and the gene deletion of FUT2 are responsible for the classical Indian Bombay phenotype.

ABO Blood-Group System↗
PubMed1997

Alternative splicing of a novel glycophorin allele GPHe(GL) generates two protein isoforms in the human erythrocyte membrane.

The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at -6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.

Alleles↗
PubMed1997

Blood group antigens Rb(a), Tr(a), and Wd(a) are located in the third ectoplasmic loop of erythroid band 3.

BACKGROUND: Rb(a), Tr(a), and Wd(a) are three low-incidence blood group antigens that have not been assigned to a particular structure of the red cell membrane. Recent genetic and serologic data suggested erythroid band 3 as a possible carrier of these three antigens. STUDY DESIGN AND METHODS: Ten band 3 gene exons that encode the membrane domain of band 3 were screened for single strand conformation polymorphism (SSCP). Exons displaying SSCP were cloned and sequenced, and the presence of the mutations was verified by restriction digestion. RESULTS: Substitutions 548 Pro-->Leu, 551 Lys-->Asn, and 557 Val-->Met, all located in the third ectoplasmic loop of band 3, were detected in the subjects with Rb(a+), Tr(a+), and Wd(a+) red cells, respectively. The presence of the Rb(a) and Wd(a) mutations was confirmed in additional carriers of these blood group antigens. Chymotryptic cleavage at Tyr 553 and Tyr 555 abolished the agglutinability of Tr(a+) and Wd(a+) cells with the corresponding antisera, further demonstrating that the epitopes are located in the third ectoplasmic loop of band 3. Similar quantities of mRNA corresponding of the two band 3 alleles, a normal pattern of red cell membrane proteins, and normal DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, disodium salt)-inhibitable sulfate flux were detected, which suggests that the mutations do not affect band 3 mRNA stability or band 3 protein expression and transport function. CONCLUSION: Wd(a) and Rb(a), and tentatively Tr(a), can be assigned to the Diego blood group system.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1995

Expression of the erythrocyte antigen Henshaw (He; MNS6): serological and immunochemical studies.

Production of murine monoclonal antibodies to the low prevalence MNS antigen Henshaw (He; MNS6) has enabled more detailed study of this antigen. Using these directly hemagglutinating anti-He, red blood cells (RBCs) from 1695 people of African origin were screened in the USA and England. The prevalence of He+ samples among these donors was 2.1%. In Natal, blood samples from 1218 black donors were screened with rabbit anti-He. The prevalence of He+ donors in this population was 7.0%. Immunoblotting confirmed that the He antigen is carried on an erythrocyte membrane component with a molecular mass that is indistinguishable from glycophorin B. Hemagglutination and immunoblotting demonstrated that ten of 56 He+ samples tested more extensively had a reduced expression of the He antigen. The majority of He+ RBCs were S+; those He+ RBC samples that were S-s+ more frequently had a weakened expression of He.

Antibodies, Monoclonal↗
PubMed1993

ERIK, a low-frequency red cell antigen of the MNS blood group system associated with Sta.

A new low-frequency red cell antigen, ERIK (MNS37), is associated with the Sta antigen of the MNS system. Four ERIK+ propositi have been identified: all are St(a+). Thirteen other St(a+) samples, including one of the Mz type, and over 200 St(a-) samples were ERIK-. In two of the propositi ERIK is associated with an abnormal trypsin-resistant M antigen; in the others it is associated with N, which is shown to be expressed weakly in one family. Immunoblotting with an antibody to an epitope common to glycophorin A (GPA) and glycophorin B (GPB) gave identical results with St(a+) ERIK+ and St(a+) ERIK- cells, revealing GPA, GPB, an abnormal structure GPSta and aggregates of these components. In the propositi with trypsin-resistant M, GPSta carries the unusual M antigen, whereas in the M-N+ERIK+ individual analysed by immunoblotting GPSta carries N. Immunoblotting with anti-Sta and anti-ERIK showed that Sta is located on GPSta but that ERIK is located on GPA, presumably the GPA molecule encoded by the GYPA gene contiguous to the gene encoding GPSta.

Adult↗
PubMed1993

STEM, a new low-frequency Rh antigen associated with the e-variant phenotypes hrS-(Rh: -18, -19) and hrB-(Rh: -31, -34).

The new low-frequency antigen STEM was identified when a Cape coloured woman with an unknown antibody in her plasma (which agglutinated the red cells of her husband and a minority of other individuals) gave birth to a baby suffering mildly from haemolytic disease of the new-born. Most, but not all, examples of anti-STEM distinguish different strengths of STEM antigen on the red cells of different people; the different strengths are inherited. Family studies established that STEM was inherited as a Mendelian dominant character. STEM subdivides hrS-(Rh: -18, -19) and hrB- (Rh: -31, -34) red cells into two types: STEM+ and STEM-. The manually calculated lod score for STEM being associated with the Rh system is 3.91 and LIPED calculated lod score 4.35. The International Society of Blood Transfusion has allocated STEM the Rh number 49.

Female↗
PubMed1991

Rh33 in two of three German siblings with D+ C+ c+ E- e+ red cells.

The proposita in a German family of three siblings has D+ C+ c+ E- e+ f+ Rh: -17,19,33,34 red cells with weak C, e, f, Rh19, and Rh34 and stronger-than-usual Rh33 expression. One sibling has D+ C+ c+ E- e+ f+ Rh:17,19,33,34 red cells with weak f and ordinary-strength Rh33, and the other sibling has D+ C+ c+ E- e+ f+ Rh:17,19,-33,34 red cells. In the absence of any further family members, the proposita's unusual phenotype suggests that she has an RoHar haplotype and a "new" Rh haplotype, provisionally named R1Lisa, that encodes Rh33, normal-strength D, weak C, weak or nondemonstrable e, Rh19, and Rh34, but not Rh17. Her Rh:33 sibling may have R1 and RoHar and her Rh:-33 sibling R1 and r haplotypes.

Adult↗