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.

Cardiac complications of systemic sclerosis.

The majority of patients with SSc are believed to have subclinical primary cardiac involvement. Overt cardiac manifestations of SSc are associated with poor prognosis and can be difficult to manage. Primary myocardial disease, i.e. without systemic or pulmonary hypertension and without significant pulmonary or renal disease, is postulated to be due to microvascular ischaemia. Undetected early cardiac manifestations can progress silently to myocardial fibrosis. Symptoms may manifest without warning and can rapidly lead to arrhythmia and left and right heart dysfunction and failure. Of the currently practical screening methods, annual echocardiography and/or evaluation of N-terminal portion of pro-B-type natriuretic peptide concentrations should therefore be employed in SSc patients, in order to anticipate the development of cardiac symptoms. Although there is limited evidence in respect of specific therapeutic options, treatment of early abnormalities with calcium channel blockers and angiotensin-converting enzyme inhibitors may improve myocardial perfusion and function, while standard management of overt cardiac disease is equally appropriate in the SSc population. However, it remains to be seen if early intervention can limit the progression of these life-threatening complications.

Pulmonary arterial hypertension: the most devastating vascular complication of systemic sclerosis.

Pulmonary arterial hypertension (PAH) is a devastating vascular complication of a number of CTDs. In patients with SSc, PAH has a dramatic impact on prognosis and survival and is the single most common cause of disease-related death.Yearly echocardiographic screening for PAH is recommended in patients with SSc. If suspected, confirmation of PAH diagnosis by right heart catheterization is necessary. Treatment goals for patients with PAH associated with SSc (PAH-SSc) aim to slow disease progression and improve quality of life. Some measures used to gauge the effect of treatment in patients with PAH-SSc remain to be fully validated; the 6-min walk distance, for example, is a simple and reproducible means of assessing exercise capacity, but there exists a need to understand what constitutes a clinically relevant change in this specific patient population. Currently, pharmacological intervention in PAH-SSc may target one or more of three pathophysiological pathways in PAH. The prostacyclin analogue epoprostenol has been shown to improve exercise capacity and haemodynamics in PAH-SSc patients and similar data are available from smaller studies on trepostinil and iloprost. The dual endothelin receptor antagonist bosentan has been shown to improve exercise capacity and haemodynamics in PAH-SSc, and similar data have been obtained in small numbers of patients treated with the endothelin receptor A antagonists sitaxsentan and ambrisentan. Impaired production of nitric oxide may be addressed by inhibiting phosphodiesterase type-5 with sildenafil or possibly tadalafil. Combinations of multiple targeted therapies may be beneficial to this patient population.

The Redelberger antigen: a family study, a family story.

The Redelberger antigen (Rba) was first discovered in 1974 on the RBCs of a blood donor who was an employee of the Community Blood Center in Dayton, Ohio. The discovery was made as a result of the investigation of a reagent contamination problem. Two examples of the Rba antigen were subsequently identified in the United Kingdom,but no "new"examples have been identified in the United States or Europe. Anti-Rba is a commonly occurring antibody, often found in combination with other antibody specificities, especially in combination with other antibodies to low-incidence antigens.

Consensus statement concerning cardiotoxicity occurring during haematopoietic stem cell transplantation in the treatment of autoimmune diseases, with special reference to systemic sclerosis and multiple sclerosis.

Autologous haematopoietic stem cell transplantation is now a feasible and effective treatment for selected patients with severe autoimmune diseases. Worldwide, over 650 patients have been transplanted in the context of phase I and II clinical trials. The results are encouraging enough to begin randomised phase III trials. However, as predicted, significant transplant-related morbidity and mortality have been observed. This is primarily due to complications related to either the stage of the disease at transplant or due to infections. The number of deaths related to cardiac toxicity is low. However, caution is required when cyclophosphamide or anthracyclines such as mitoxantrone are used in patients with a possible underlying heart damage, for example, systemic sclerosis patients. In November 2002, a meeting was held in Florence, bringing together a number of experts in various fields, including rheumatology, cardiology, neurology, pharmacology and transplantation medicine. The object of the meeting was to analyse existing data, both published or available, in the European Group for Blood and Marrow Transplantation autoimmune disease database, and to propose a safe approach to such patients. A full cardiological assessment before and during the transplant emerged as the major recommendation.

Computer-generated attenuation correction does not improve the accuracy of myocardial perfusion scintigraphy.

A short study was performed to determine if it is possible to increase the accuracy of thallium-201 (201T1) single photon emission tomographic myocardial perfusion imaging using computer-generated ('Chang') attenuation correction. The stress and rest myocardial perfusion studies from 22 patients with suspected or known ischaemic heart disease were reconstructed with and without "Chang' attenuation correction. For all patients, the scintigraphy results were compared with those of coronary angiography. Attenuation correction improved the accuracy of 201T1 myocardial perfusion imaging for defining myocardial ischaemia or infarction in 8% of coronary artery territories (23% of patients), but it was worse in 5% of coronary artery territories (14% of patients). These changes were not significant (McNemar's test). Therefore, computer-generated 'Chang' attenuation correction does not appear to improve the accuracy of myocardial perfusion scintigraphy. It is important that all techniques suggested to improve the accuracy of clinical images should be tested on patients before being widely used.

Screening for pulmonary arterial hypertension in systemic sclerosis.

The onset and progression of pulmonary arterial hypertension (PAH) in patients with systemic sclerosis (SSc) can be particularly aggressive; however, effective treatments are available. Therefore, early identification of patients with suspected PAH, confirmation of diagnosis, and intervention is essential. PAH may be challenging to diagnose in its earliest stages, particularly in populations that have multiple causes of breathlessness, and, therefore, screening is required. The optimal screening tools and methodology are, as yet, unknown, and this is confounded by a lack of consensus over which patients to screen. Current practice favours annual screening of all SSc patients using Doppler echocardiography to detect elevated right heart pressures. This will typically identify most patients with the various forms of pulmonary hypertension found in SSc. The optimum thresholds for Doppler echocardiography are still subject to investigation, especially for patients with mild pulmonary hypertension, and this technique may, therefore, yield a significant number of false-positives and a currently unknown number of false-negatives. Confirmatory right heart catheterisation remains necessary in all suspected cases. Further research is needed to identify the optimal tools and the screening approach with greatest specificity and selectivity.

Effects of enzyme replacement therapy on the cardiomyopathy of Anderson-Fabry disease: a randomised, double-blind, placebo-controlled clinical trial of agalsidase alfa.

BACKGROUND: Anderson-Fabry disease is an X-linked glycosphingolipid storage disorder caused by deficient activity of the lysosomal enzyme alpha-galactosidase A. This leads to a progressive accumulation of globotriaosylceramide (Gb(3)) in the lysosomes of cells throughout the body that ultimately results in premature death from renal, cardiac or cerebrovascular complications. Until recently, there was no effective therapy available for this disease. The present study was designed to assess the safety and efficacy of enzyme replacement therapy with agalsidase alfa on the cardiac manifestations of Anderson-Fabry disease. METHOD: The effects of therapy with agalsidase alfa on cardiac structure and function were assessed in a randomised, double-blind, placebo-controlled study of 15 adult male patients with Anderson-Fabry disease. The following parameters were measured at baseline and 6 months: left ventricular mass, QRS duration and levels of Gb(3) in cardiac tissue, urine sediment and plasma. After 6 months of the randomised trial patients were enrolled in a 2-year open-label extension study. RESULTS: Left ventricular mass, as measured by MRI, was significantly reduced following 6 months of treatment with agalsidase alfa compared with placebo (p = 0.041). A mean 20% reduction in myocardial Gb(3) content as assessed by serial transvenous endomyocardial biopsies was demonstrated over the 6 months of enzyme replacement compared to a mean 10% increase in patients receiving placebo (p = 0.42) CONCLUSION: Enzyme replacement therapy with agalsidase alfa resulted in regression of the hypertrophic cardiomyopathy associated with Anderson-Fabry disease.

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.

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.

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 Diego blood group locus is located on chromosome 17q.

The Diego blood group locus (DI) is tightly linked to the erythrocyte surface protein band 3 locus (EPB3) with z = 5.42 at theta = 0.00 for combined paternal and maternal meioses. Looser linkage between DI and D17S41 (z = 3.14 at theta = 0.09) for combined paternal and maternal meioses was also established. Taken together, these results indicate that the Diego blood group locus is located on the long arm of chromosome 17.

Assignment of the Auberger red cell antigen polymorphism to the Lutheran blood group system: genetic justification.

Family studies have provided the final piece of evidence for the assignment of Auberger to the Lutheran blood group system. Lods derived from combined paternal and maternal meioses (zeta = 10.83 at theta = 0.00) strongly support the contention that a single gene controls the expression of both Au and LU antigens. Consequently, the International Society of Blood Transfusion Working Party on Terminology has designated Aua and Aub as LU18 and LU19, respectively.

Assignment of the YT blood group locus to chromosome 7q.

The antithetical antigens YT1 and YT2 constitute the YT blood group system (International Society of Blood Transfusion system number 11). Despite being serologically well defined, the YT blood group locus (YT) has not secured a chromosomal location. In our report, peak lods of 3.61 at theta = 0.00 for YT:COL1A2 and of 3.31 at theta = 0.00 for YT:D7S13 allow us to assign YT to the long arm of chromosome 7.

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.

A "new" low-incidence red cell antigen, LOCR, associated with altered expression of Rh antigens.

BACKGROUND: Mild cases of hemolytic disease of the newborn were being studied when it was recognized that the propositi's red cells carried a novel antigen. STUDY DESIGN AND METHODS: Serologic and genetic studies of a new low-incidence antigen, LOCR (International Society of Blood Transfusion series number 700.53), were performed. RESULTS: The antigen is associated with altered expression of the Rh antigen c in two unrelated families and in a third proposita and with an altered expression of e in a fourth proposita. The lods for the gene controlling LOCR relative to Rh are 2.107 at theta = 0.00; that is, they fall short of the requirement for inclusion of the antigen in the Rh blood group system. LOCR is excluded from the ABO, MNS, Lutheran, Kell, Duffy, Kidd, Xg, Chido/Rodgers, Kx, and Gerbich blood group systems. Genetic evidence suggests it is not part of the Yt, Colton, or LW systems. CONCLUSION: Serologic evidence that LOCR is associated with altered expression of c or e strongly suggests that LOCR is part of the Rh blood group system. However, the genetic evidence falls short of the level required for system assignment.

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.

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.