Genotyping an immunodeficiency causing c.1624-11G>A ZAP70 mutation in Canadian Mennonites.

BACKGROUND: Primary immunodeficiency is a life-threatening genetic disease that appeared to have an increased incidence in Manitoba Mennonites. Determining the genetic basis of this immunodeficiency was an essential step for providing early and appropriate medical intervention. METHODS: Initially, DNA from probands affected with primary immunodeficiency and their family members was assessed for linkage to genes previously associated with immunodeficiency. Candidate genes were sequenced to identify the causative mutation. The frequency of the mutation among first and second degree relatives, as well as apparently unrelated community members was analyzed using a PCR-based assay. RESULTS: A previously described c.1624-11G>A mutation in ZAP70 was identified as the causative mutation in all affected probands that were analyzed. Among 125 study participants of Mennonite descent, 79 genotyped as normal, 39 were carriers and seven were affected. None of 115 non-Mennonite random individuals carried the mutation, whereas one of ten random DNA samples from individuals who self-identified as Mennonite was a carrier. CONCLUSIONS: In collaboration with the target community, we have developed a robust screening test for determining ZAP70 genotype. Early identification of affected individuals has provided an opportunity for timely clinical intervention, while carrier identification has allowed for genetic counselling of at risk couples.

International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012).

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

Refinement of the hereditary xerocytosis locus on chromosome 16q in a large Canadian kindred.

The hereditary stomatocytoses are a group of heterogeneous conditions associated with chronic red cell hemolysis for which the causative genetic mutations are not known. We investigated 137 members of a large Canadian kindred with phenotypic findings consistent with hereditary xerocytosis, one of the most common stomatocytosis syndromes. The objectives of this study were to characterize the clinical hallmarks of the hemolytic process, and to define the chromosomal region carrying the disease locus. The mode of inheritance was autosomal dominant. Affected family members had a well-compensated hemolysis, associated with an elevated MCHC, decreased osmotic fragility, decreased haptoglobin, and indirect hyperbilirubinemia. Cholelithiasis and progressive iron loading were common, despite normal hemoglobin levels. Quantitative erythrocyte morphologic evaluation revealed increased schistocytes, target cells, reticulocytes, and eccentrocytes in affected individuals; stomatocytes were not increased. Genetic linkage analysis confirmed the localization of the disease phenotype to chromosome 16q, and refined the candidate region to 16q24.2-16qter, a 2.4 million base pair interval containing 51 known or predicted genes.

Molecular basis of succinylcholine sensitivity in a prairie Hutterite kindred and genetic characterization of the region containing the BCHE gene.

The tetrameric glycoprotein butyrylcholinesterase (BChE; EC 3.1.1.8) is one of two enzymes that hydrolyze choline esters. The controlling gene (BCHE) is comprised of four coding exons and is located on chromosome 3q26. Based on BChE activity measurements in the presence and absence of dibucaine, usual (designated U) and atypical (designated A) gene products have been distinguished. Homozygotes for the A gene product are at risk for prolonged apnea following exposure to the surgical anesthetics succinylcholine or mivacurium. In this report, we detail biochemical and molecular investigations of succinylcholine sensitivity in a prairie Hutterite kindred. Our results establish that BChE activities in the family members are impacted by two distinct BCHE mutations, namely, c.209A>G p. D70G and c.1615G>A p. A539T. However, homozygotes for the c.209A>G mutation (i.e., atypical or A) are the only individuals whose BChE activity could lead to adverse reactions to succinylcholine. Interestingly, haplotype analysis of the chromosomal region containing BCHE indicates that the c.209A>G mutation is carried on a unique haplotype, suggesting that it was likely introduced into the population only once. Conversely, the c.1615G>A mutation is carried on various haplotypes and was likely introduced into the population more than once.

Incorporation of fatty acids into phosphatidylcholine is reduced during storage of human erythrocytes: evidence for distinct lysophosphatidylcholine acyltransferases.

The incorporation of [1-14C]palmitic or [1-14C]oleic acid into phosphatidylcholine and the effect on blood group antigen expression were examined in human erythrocytes stored at 4 degrees C for 0-3 weeks. Blood drawn into EDTA was obtained by venepuncture from healthy volunteers. A 50% suspension of washed erythrocytes was incubated in buffer containing [1-14C]fatty acid for up to 60 min at 37 degrees C with moderate shaking. Phosphatidylcholine was extracted and analyzed for uptake of radiolabelled fatty acid and phospholipid phosphorus content. Incorporation of [1-14C]palmitic or [1-14C]oleic acid into phosphatidylcholine was reduced during storage. The mechanism for the reduction in radiolabelled fatty acid incorporation into phosphatidylcholine was a 64% (p < 0.05) reduction in membrane phospholipase A2 activity. Although human erythrocyte membranes isolated from freshly drawn blood are capable of reacylating lysophosphatidylcholine to phosphatidylcholine, with storage, a markedly different substrate preference between palmitoyl-Coenzyme A and oleoyl-Coenzyme A was observed. Lysophosphatidylcholine acyltransferase activity assayed with oleoyl-Coenzyme A was unaltered with storage. In contrast, lysophosphatidylcholine acyltransferase activity assayed with palmitoyl-Coenzyme A was elevated 5.5-fold (p < 0.05). Despite these changes, storage of erythrocytes for up to 3 weeks did not result in altered expression of the various blood group antigens investigated. We conclude that the incorporation of palmitate and oleate into phosphatidylcholine is dramatically reduced during storage of human erythrocytes. The observed differential in vitro substrate utilization suggests that distinct acyltransferases are involved in the acylation of lysophosphatidylcholine to phosphatidylcholine in human erythrocytes.

Synthesis of a triply phosphorylated pentapeptide from human tau-protein.

Two different strategies for the synthesis of a triply phosphorylated pentapeptide are described. In both cases a monophosphorylated selectively N-deprotected tripeptide is employed as C-terminal fragment. Coupling of this building block with a C-terminally unmasked bis-phosphorylated seryl-dipeptide unexpectedly failed due to decomposition of this peptide upon activation with different coupling reagents. Instead stepwise N-terminal elongation of the peptide chain with serine derivatives and subsequent O-phosphorylation of the serine OH-groups was successful. These results indicate that assembly of multiply phosphorylated peptides from preformed multiply phosphorylated phosphopeptide building blocks in general may be problematic and that a stepwise elongation of the amino acid chain may be preferable.

An amino acid substitution in the putative second extracellular loop of RBC band 3 accounts for the Froese blood group polymorphism.

BACKGROUND: The low incidence RBC antigen Fr(a) has been excluded from 17 of the 25 established blood group systems. Previous genetic analysis assigned the gene controlling Fr(a) expression to the same chromosomal region as the solute carrier family 4, anion exchanger member 1 gene (SLC4A1). Because SLC4A1 encodes RBC band 3 and controls the expression of Diego blood group system antigens, the possible relationship of Fr(a) to the Diego blood group system was investigated by molecular analysis of SLC4A1. STUDY DESIGN AND METHODS: Blood samples were obtained from the members of two unrelated Mennonite kindreds segregating for Fr(a). DNA was extracted, amplified by PCR using intronic primer sets flanking exons 11-20 of SLC4A1, and screened by single-strand conformation polymorphism (SSCP) analysis. Those exons displaying SSCPs were subjected to DNA sequence analysis. RESULTS: An exon 13 SSCP mobility shift was observed in the DNA from all Fr(a+) persons that was not seen in the DNA from Fr(a-) family members or control subjects. Linkage between the exon 13 SSCP and FR:(a) was established, with peak lods = 3.62 at theta = 0.00 for combined paternal and maternal meioses. DNA sequencing revealed a GAG --> AAG mutation that underlies a Glu480Lys substitution in RBC band 3. CONCLUSIONS: A point mutation in exon 13 of SLC4A1 accounting for a Glu480Lys substitution in band 3 controls Fr(a) expression. On the basis of these our results, the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens has assigned Fr(a) to the Diego blood group system, with the designation DI20.

Confirmation of the assignment of the Dombrock blood group locus (DO) to chromosome 12p: narrowing the boundaries to 12p12.3-p13.2.

BACKGROUND AND OBJECTIVES: Antigens belonging to the Dombrock blood group system have been well characterized serologically. Despite being reasonably polymorphic, chromosomal localization of the gene controlling Dombrock expression (DO) has proved difficult. In the past, DO had been provisionally assigned to chromosomes 1 and 4; more recently, DO was provisionally assigned to chromosome 12. The current study was undertaken in an attempt to confirm or refute this latest assignment. MATERIALS AND METHODS: DNA from a series of families segregating for DO was amplified by polymerase chain reaction and analyzed for trinucleotide or tetranucleotide repeat polymorphisms of four anonymous DNA markers (D12S1042, D12S373, D12S391, D12S372) and of the von Willebrand factor gene (VWF). Lods for or against linkage were determined between DO and each marker. RESULTS: DO is linked to all five markers. The closest linkage was established between DO and D12S373, and DO and D12S391, with peak lods for combined paternal and maternal meioses of 7.16 at peak recombination fraction (theta) = 0.08 and 7.69 at theta = 0.08, respectively. CONCLUSIONS: We have confirmed the assignment of the Dombrock blood group locus to chromosome 12p and have refined its regional localization to 12p12.3-p13.2.

Amino acid substitutions in human erythroid protein band 3 account for the low-incidence antigens NFLD and BOW.

BACKGROUND: The low-incidence red cell antigens NFLD (700.37) and BOW (700.46) were first described in 1984 and 1988, respectively. Recent investigations showed that antigens of the Diego blood group system (including a number of low-incidence antigens) are coded by SLC4A1 (solute carrier family 4, anion exchanger member 1 gene). Among these newly characterized Diego system antigens is Wu (designated DI9). Because a serologic relationship among Wu, NFLD, and BOW has been established, a series of genetic and molecular investigations of SLC4A1 in relation to NFLD and BOW were undertaken. STUDY DESIGN AND METHODS: By the use of exon-specific primers, single-strand conformational polymorphism (SSCP) analysis of SLC4A1 was performed on DNA isolated from an NFLD+ person from Japan, from the members of a Canadian kindred segregating for NFLD, and from two unrelated BOW+ persons. Exons displaying SSCPs were subjected to genetic linkage analysis (for NFLD only) and DNA sequencing. RESULTS: SSCPs in DNA amplified from exons 12 and 14 of SLC4A1 were observed for all NFLD+ subjects. Linkage between each of these polymorphisms and NFLD was established with peak lods = 4.82 at theta = 0.00 for combined paternal and maternal meiosis. DNA sequencing of exons 12 and 14 of SLC4A1 from NFLD+ persons identified A-->T and C-->G mutations that underlie Glu429Asp and Pro561Ala substitutions in human erythroid band 3 protein (band 3). DNA from the two unrelated BOW+ persons only exhibited an SSCP in exon 14 of SLC4A1. Subsequent DNA sequencing revealed a C-->T mutation that accounts for a Pro561Ser substitution in band 3. CONCLUSION: SLC4A1 codes for the low-incidence red cell antigens NFLD and BOW. In light of these findings, both antigens have been assigned to the Diego blood group system.

Distinctive Swann blood group genotypes: molecular investigations.

BACKGROUND AND OBJECTIVES: Phenotypically, Sw(a+) erythrocytes have been classified as either 700:4,41 or 700:4,-41. Since anti-700.4, in particular, and sometimes anti-700.41 are contained in reagents defining other low-incidence antigens that are members of the Diego blood group system, we undertook the current investigation in an attempt to establish whether or not Swann antigens are also Diego system members. MATERIALS AND METHODS: DNA from the members of three unrelated kindreds whose red cells type as Sw(a+) was isolated and analyzed for variation in SLC4A1 (solute carrier family, anion exchanger member 1 gene) by single-strand conformational polymorphism (SSCP) and DNA sequence analyses. RESULTS: Polymerase chain reaction-amplified exon 16 SLC4A1 products from the DNA of all Sw(a+) individuals displayed a mobility shift by SSCP. A similar mobility shift was not observed in the DNA from Sw(a-) family members or in the amplified DNA from control individuals. DNA sequencing revealed different mutations, CGG-->CAG and CGG-->TGG, that result in Arg646Gln and Arg646Trp substitutions in erythroid protein band 3, respectively. CONCLUSION: Through genotypic analyses, we have characterized two point mutations related to the Swann blood group. The possible relationship between the resultant amino acid substitutions and the expression of Swann antigens has been discussed.

The ELO blood group polymorphism is located in the putative first extracellular loop of human erythrocyte band 3.

BACKGROUND AND OBJECTIVES: The low incidence red cell antigen ELO (700.51) has been excluded from 13 of 23 established blood group systems. Information relative to the Diego system has not been reported. To investigate a possible association between ELO and the gene controlling Diego blood group polymorphisms, we undertook molecular studies of AE1 (anion exchanger 1 gene). MATERIALS AND METHODS: DNA from a family segregating for ELO and from the original ELO proposita was amplified by PCR using intronic primers flanking exons 11-20 of AE1. Subsequently, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were conducted. RESULTS: We have observed an exon 12 SSCP in all ELO+ individuals tested. This SSCP is the result of a C-->T mutation in exon 12 of AE1 which leads to an Arg432-->Trp amino acid substitution in the putative first extracellular loop of band 3 and thereby accounts for the Diego blood group system polymorphism known as ELO. CONCLUSIONS: In light of these findings, the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens has designated ELO as a member of the Diego blood group system (010008 or D18) and rendered the previous numerical designation (700.51) obsolete.

A Gly565-->Ala substitution in human erythroid band 3 accounts for the Wu blood group polymorphism.

BACKGROUND: Reports published in 1976 and 1980 described the low-incidence red cell antigen Wu. Distinction of Wu from all other known low-incidence antigens and from the ABO, Rh, Lutheran, Duffy, Kidd, P, and X-linked blood group systems allowed Wu to be placed in the International Society of Blood Transfusion's 700 series, designated as 700013. Recently, a blood donor apparently homozygous for Wu has been identified. This report documents the serologic and molecular findings of samples from this individual and the members of his family. STUDY DESIGN AND METHODS: Blood samples from 26 members of a kindred of Dutch descent segregating for Wu were collected and analyzed. Red cells were subjected to titration and enzymatic tests, while DNA was analyzed by polyacrylamide gel electrophoresis for single-strand conformational polymorphism (SSCP) and nucleotide differences by DNA sequencing. RESULTS: Serologic investigations conducted on red cells of the propositus and two of his siblings consistently revealed higher titers with various sera containing anti-Wu than did cells from their parents or children. Treatment of intact red cells with alpha-chymotrypsin completely abolished Wu recognition. Because erythroid band 3 is cleaved by alpha-chymotrypsin, the possible relationship between Wu and AE1 (the gene controlling erythroid band 3 expression) was investigated by molecular methods. SSCP analysis of DNA revealed that all Wu+ family members exhibited a mobility shift in exon 14 of AE1. The nature of the SSCP was defined by DNA sequencing as a G-->C mutation that resulted in a Gly565-->Ala substitution in human erythroid band 3. CONCLUSIONS: Three members of the kindred are homozygous for the low-incidence red cell antigen Wu. A G-->C mutation in AE1 gives rise to a Gly565-->Ala substitution in band 3, thereby accounting for the Wu red cell polymorphism. In light of these findings, the International Society of Blood Transfusion Working Party has provisionally assigned Wu to the Diego blood group system (designated 010009 or D19).