A simple screening assay for the most common JK*0 alleles revealed compound heterozygosity in Jk(a-b-) probands from Guam.

The Jk(a-b-) phenotype results from alterations in the JK gene and is characterized by absence of the RBC urea transporter in the cell membrane. The frequency of Jk(a-b-) varies among populations,but this phenotype is most commonly found in people of Polynesian and Finnish descent. Although rare, Jk(a-b-) individuals present a clinical challenge because anti-Jk3 is produced readily in response to transfusion and pregnancy, and Jk(a-b-) blood is not routinely available. Identification of Jk(a-b-) patients and donors is most often performed serologically. However, ten JK*0 alleles have been identified, and this information can be used in DNA-based typing. We selected five JK*0 alleles that had been encountered by our reference laboratory in two or more samples from unrelated individuals and designed an allele-specific primer PCR assay for use as an initial screening tool. After in-house validation,we tested genomic DNA from a family: a mother and her two sons referred to us for genetic investigation of their Jk(a-b-)phenotypes. Two different nucleotide substitutions, -1g>a in intron 5 (IVS5) and 956C>T in exon 10, originally associated with Polynesian and Indian/African populations respectively, were identified in the family. The mother and one son were compound heterozygotes, and the second son was homozygous for IVS5-1g>a. We conclude that the effort to design and validate such a screening assay was cost-efficient when compared with DNA sequencing costs. Furthermore, selection of the more common JK*0 mutations was a practical approach that resulted in rapid identification of the genetic bases behind the Jk(a-b-) phenotypes in this unusual family. Although an obvious target for eventual inclusion into high-throughput genotyping platforms for clinical diagnostic services, current systems are very limited. Our approach provides a simple and inexpensive method for the identification of these rare alleles.

Fragmentation of riverine systems: the genetic effects of dams on bull trout (Salvelinus confluentus) in the Clark Fork River system.

Migratory bull trout (Salvelinus confluentus) historically spawned in tributaries of the Clark Fork River, Montana and inhabited Lake Pend Oreille as subadult and adult fish. However, in 1952 Cabinet Gorge Dam was constructed without fish passage facilities disrupting the connectivity of this system. Since the construction of this dam, bull trout populations in upstream tributaries have been in decline. Each year adult bull trout return to the base of Cabinet Gorge Dam when most migratory bull trout begin their spawning migration. However, the origin of these fish is uncertain. We used eight microsatellite loci to compare bull trout collected at the base of Cabinet Gorge Dam to fish sampled from both above and further downstream from the dam. Our data indicate that Cabinet Gorge bull trout are most likely individuals that hatched in above-dam tributaries, reared in Lake Pend Oreille, and could not return to their natal tributaries to spawn. This suggests that the risk of outbreeding depression associated with passing adults over dams in the Clark Fork system is minimal compared to the potential genetic and demographic benefits to populations located above the dams.

Gene-centromere mapping of 312 loci in pink salmon by half-tetrad analysis.

We estimated recombination rates between 312 loci and their centromeres in gynogenetic diploid pink salmon (Oncorhynchus gorbuscha) that we produced by initiating development with irradiated sperm and blocking the maternal second meiotic division. Amplified fragment length polymorphisms (AFLPs) were significantly more centromeric than loci identified by three other techniques (allozymes, microsatellites, and PCR using primer sequences from interspersed nuclear elements). The near absence of AFLPs in distal regions could limit their utility in constructing linkage maps. A large proportion of loci had frequency of second division segregation (y) values approaching 1.0, indicating near complete crossover interference on many chromosome arms. As predicted from models of chromosomal evolution in salmonids based upon results with allozyme loci, all duplicated microsatellite loci that shared alleles (isoloci) had y values of nearly 1.0.

Inheritance of nuclear DNA markers in gynogenetic haploid pink salmon.

We describe the inheritance of 460 PCR-based loci in the polyploid-derived pink salmon (Oncorhynchus gorbuscha) genome using gynogenetic haploid embryos. We detected a length polymorphism in a growth hormone gene (GH-2) intron that is caused by an 81 bp insertion homologous to the 3' end of the salmonid short interspersed repetitive element (SINE) SmaI. Such insertion polymorphisms within species bring into question the use of SINEs as phylogenetic markers. We confirmed that a microsatellite locus encodes a PCR-null allele that is responsible for an apparent deficit of heterozygotes in a population sample from Prince William Sound. Another set of microsatellite primers amplified alleles of the same molecular weight from both loci of a duplicated pair. In our analysis of several PCR-based multilocus techniques, we failed to detect evidence of comigrating fragments produced by duplicated loci. Segregation analysis of PCR-based markers using gynogenetic haploid embryos ensures that the interpretation of molecular variation is not complicated by heterozygosity, diploidy, or gene duplication. We urge investigators to test the inheritance of polymorphisms in salmonids prior to using them to measure genetic variation.

EDTA/glycine-acid versus chloroquine diphosphate treatment for stripping Bg antigens from red blood cells.

EDTA/glycine-acid (EGA) has been reported to remove IgG-bound antibodies from red blood cells (RBCs) and to denature Kell system and Era antigens. EGA-treated RBCs were tested in parallel with chloroquine diphosphate (CDP)-treated RBCs to evaluate whether EGA would remove Bg antigens from RBCs as efficiently as CDP. Fifty-seven serum/plasma samples containing known Bg antibodies were tested with untreated Bg+ RBCs, EGA-treated Bg+ RBCs, and CDP-treated Bg+ RBCs by an indirect antiglobulin test (IAT), using a low-ionic-strength additive solution and murine monoclonal polyspecific antiglobulin reagent. Of 57 samples, 40 (22 anti-Bga, 17 anti-Bgb, and 1 Bg-related) were nonreactive by IAT with EGA-treated RBCs and CDP-treated RBCs, 14 (7 anti-Bga, 4 anti-Bgb, and 3 Bg-related) were nonreactive by IAT only with EGA-treated RBCs, none were nonreactive in IAT with only CDP-treated RBCs, and 3 (anti-Bga) were still reactive by IAT with EGA-treated RBCs and CDP-treated RBCs. Therefore, EGA strips Bg antigens from RBCs. Our results indicate EGA treatment is more efficient and requires less time (1-2 minutes) to perform than the CDP procedure (30-120 minutes) for removal of Bg antigens from RBCs.

Comparison of affinity column technology and LISS tube tests.

Proteins G and A coated on agarose have been extensively used in affinity chromatography. Protein G will bind to all four subclasses of human IgG and protein A to the subclasses IgG1, IgG2, and IgG4. This IgG binding ability of protein G and protein A has been used in a red cell affinity column technology developed for the detection and identification of IgG red cell antibodies. When serum or plasma is incubated in a microcolumn with red blood cells (RBCs) that express the appropriate antigens, the antibodies become attached to the RBC surface. When the microcolumns are centrifuged, the RBCs pass through a viscous barrier into an active matrix containing proteins G and A. Positive tests adhere at the top of the gel and negative tests pass through, settling to the bottom. This study was undertaken to compare affinity column technology with low-ionic saline solution (LISS) tube tests in a reference laboratory setting. Over a 1-year period, 314 samples were tested in parallel by affinity column technology and by LISS tube technique. Both methods detected antibodies directed at common RBC antigens, high-incidence and low-incidence RBC antigens, and warm-reacting autoantibodies. IgM antibodies were not detected by affinity column technology. Affinity column technology compares favorably with the LISS tube technique for IgG antibody detection and identification.

GIL: a red cell antigen of very high frequency.

A new high-frequency red cell antigen has been identified and named GIL. GIL differs from all high-frequency antigens included in the International Society of Blood Transfusion classification. There is very little family information and GIL has not been shown to be an inherited character. Five women with anti-GIL have been found. All had been pregnant at least twice. Red blood cells of two of the babies gave positive direct antiglobulin tests, but there were no clinical signs of hemolytic disease. Anti-GIL may have been responsible for a hemolytic transfusion reaction and results of monocyte monolayer assays of two of the anti-GIL suggested a potential to cause destruction of transfused GIL+ RBCs.

SINE sequences detect DNA fingerprints in salmonid fishes.

DNA probes homologous to two previously described salmonid short interspersed nuclear elements (SINEs) detected DNA fingerprint patterns in 14 species of salmonid fishes. The probes showed more homology to some species than to others and little homology to three nonsalmonid fishes. The DNA fingerprint patterns derived from the SINE probes are individual-specific and inherited in a Mendelian manner. Probes derived from different regions of the same SINE detect only partially overlapping banding patterns, reflecting a more complex SINE structure than has been previously reported. Like the human Alu sequence, the SINEs found in salmonids could provide useful genetic markers and primer sites for PCR-based techniques. These elements may be more desirable for some applications than traditional DNA fingerprinting probes that detect tandemly repeated arrays.

A second example of anti-Esa, an antibody to a high-incidence Cromer antigen.

A blood sample contained an antibody to a high-incidence antigen that reacted with all red blood cells (RBCs) tested by the indirect antiglobulin test (IAT). The antibody reacted with papain-, ficin-, and trypsin-treated RBCs, but not with a-chymotrypsin-treated RBCs. This pattern of reactivity suggested the possibility that the antibody was recognizing an antigen in the Cromer blood group system. Tests against RBCs deficient in decay-accelerating factor (which carries the Cromer antigens) were weakly positive. Tests with antibodies to high-incidence Cromer antigens and with RBCs lacking high-incidence Cromer antigens led to identification of the second example of anti-Esa in an Es(a-) person. The antibody was IgG1 and reacted by the IAT to a titer of 64. The monocyte monolayer assay indicated potential clinical significance of this antibody in relation to transfusion.

Anti-Holley detected in a primary immune response.

Anti-Holley (Hy) has been reported as an IgG antibody occurring in previously transfused or multiparous black patients. In this case anti- Hy was identified in a 16-year-old black, primigravida female admitted at 32 weeks gestation because of premature rupture of the membranes. On admission, her blood type was determined to be A2B, D-positive and an antibody screen was negative. A second antibody screen, performed 4 days later, was positive in all three cells. Anti-Hy was subsequently identified. The antibody was reactive at room temperature, 37 degrees C, and in the antiglobulin phase. IgG and IgM components of anti-Hy were demonstrated in the maternal serum, documenting a primary immune response. This resulted in serologic findings not previously described for anti-Hy. A direct antiglobulin test on the newborn red cells was negative and there was no clinical evidence of hemolytic disease of the newborn (HDN). A monocyte monolayer assay performed with maternal serum yielded negative results. Recent scientific information has resulted in the placement of Hy in the Dombrock blood group system. Alloantibodies to Dombrock system antigens have not been associated with severe HDN.

A 'new' low-incidence red cell antigen, WARR: unique to Native Americans?

Investigation of a mild case of hemolytic disease of the newborn has led to recognition of a 'new' low-incidence red cell antigen, WARR (ISBT No. 700.55). Data gleaned from two kindreds, both with Native American heritage, exclude WARR from the MNS, Lutheran, Lewis, Duffy, Kidd, Xg, Chido/Rodgers, Kx and Gerbich blood group systems. Serologic or genetic evidence suggests it is not part of the Kell or Yt systems.

The first reported case of anti-Dob causing an acute hemolytic transfusion reaction.

The antibodies of the Dombrock blood group system have only rarely been encountered in transfusion practice, and anti-Dob has not previously been implicated in an acute hemolytic transfusion reaction. We have encountered the first such case involving a chronically transfused black female with hemoglobin SS disease and multiple antibodies in her serum. During a previous admission for sickle cell crisis, the patient received 3 units of compatible blood with no untoward effects. Serum obtained 21 days later contained, in addition to the known antibodies, anti-S plus an unidentified antibody showing characteristics of HTLA. Blood lacking the E, K1, Fy(a), Jk(b) and S antigens was obtained, and 2 least incompatible units were transfused. While administering the second unit, the patient complained of fever and low back pain, and hemoglobinemia was detected. Anti-Dob was identified in the post-reaction samples by absorption-elution tests, and the patient was confirmed to be Do(a+b-). The first unit transfused during this hemolytic episode tested Do (b+). This case, and a similar case involving anti-Doa reported in 1986, strengthens the belief that Dombrock antibodies are clinically significant and illustrates the need for their differentiation, prior to transfusion from less clinically significant HTLA antibodies.

An anti-EnaTS detected in the serum of an MiI homozygote.

The serum of EH reacted with all red cells (RBCs) except her own, ficin- or trypsin-treated red cells, and En(a-) red cells. This reactivity defined an anti-EnaTS specificity. The red cells of the proposita typed as M-N+S-S+, Vw+Mur-Hil-Hut-Anek-Lane-, Wr(a-b+), EnaKT+. Red cells of five relatives were Vw+ and positive with her serum. Titration studies suggest that EH is genetically an MiI homozygote and that her Vw+ relatives are MiI heterozygotes. There is no history of consanguinity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting studies have agreed with the serologic observations. A variant sialoglycoprotein of faster mobility than normal glycoprotein A, but no normal glycoprotein A, was detected on her red cells. Treatment with N-glycanase did not alter the mobility, which indicated that there was no N-glycosylation of residue 26. These findings are in agreement with the reported properties of the Mi.I-specific glycoprotein A. The relatives' Vw+ red cells showed the variant sialoglycoprotein and normal glycoprotein A. EH appears to be the first reported MiI homozygote.

Molecular basis for the human erythrocyte glycophorin specifying the Miltenberger class I (MiI) phenotype.

Human glycophorin Mil (HGpMil) is a structural variant of the MNSs blood group system that specifies the Miltenberger class I phenotype. We report here the molecular basis of the HGpMil gene identified in a white family in which the first homozygote was encountered. Immunoblotting analysis showed the expression of HGpMil and HGpB but the absence of HGpA on the homozygous Mil erythrocytes. Southern blot analysis detected no gross alterations in gene structure or band intensity. Genomic sequences encompassing exons II and III of the HGpMil gene were amplified by single-copy polymerase chain reaction. Restriction digestion and direct DNA sequence analysis showed that HGpMil gene is derived from an alpha N allele of HGpA and differs from the latter in the third exon by a single nucleotide change. In HGpMil, the presence of a deoxythymidine at the second position of codon 28 (ATG) not only resulted in a methionine substitution but also altered the consensus sequence for N-glycosylation from Asn-Asp-Thr to Asn-Asp-Met. These data are consistent with the occurrence of Mil on the red blood cell membrane as a variant deficient in the asparagine-linked carbohydrate unit. Significantly, this particular point mutation lies in between the two half-sites of a direct repeat that has been implicated to facilitate the recombination events leading to several other glycophorin genes of the Miltenberger series. Based on this relatedness, we propose an untemplated nucleotide replacement resulting from a gene conversion event as the molecular basis for the origin of HGpMil gene.

A family showing inheritance of the Anton blood group antigen AnWj and independence of AnWj from Lutheran.

A 43-year-old Arab woman was found to be negative for the high incidence AnWj antigen and her serum contained anti-AnWj. Two of her seven siblings were also AnWj-negative, which provides evidence for the first time that the AnWj-negative phenotype may be an inherited character. Blood groups of the family, in which the parents of the proposita are consanguineous, show that AnWj is not part of the ABO, Rh, MNSs, Kell, Duffy, Kidd, Xg and, notably, Lutheran blood group systems and neither is it X or Y linked.