alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.

tions were normal in the three patients. The additional band present in the patients’ red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients’ erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

-Mutations of the gene encoding tissue factor pathway inhibitor (TFPI), an inhibitor of TF-induced activation of the coagulation cascade, were screened for in 130 patients and 142 healthy controls to determine whether these variants contribute to acute coronary syndromes or modify plasma TFPI levels. The following 3 new polymorphisms were identified: 384T-->C in exon IV, which does not change the corresponding amino acid (tyrosine 57); -33C-->T in intron 7 (the T/T, C/T, and C/C genotypes were found in approximately 50%, 40%, and 10% of subjects in both groups); and 874G-->A in exon IX (GTG-->ATG), which predicts a valine to methionine change (V264M) in the carboxy-terminus tail of TFPI. The V264M polymorphism was found in 9.2% of the cases and 4.9% of the controls; the associated odds ratio (OR) for acute coronary syndromes was 2.0 (95% confidence interval [CI], 0.7 to 5.1). The OR increased to 3.6 (95% CI, 0.8 to 15.7) and 3.2 (95% CI, 0.9 to 11.8) in nonsmokers and patients without other risk factors, respectively. The possible link between the V264M polymorphism and coronary heart disease was checked in a large case-control study of myocardial infarction (Etude Cas-Témoins de l'Infarctus du Myocarde [the ECTIM Study]). The results showed no link between the V264M polymorphism and coronary syndromes. Interestingly, however, 5 patients heterozygous for the V264M polymorphism had significantly lower plasma TFPI levels than did 13 patients with the most common genotype. Although our present results do not support an association between TFPI polymorphisms and acute coronary syndromes, the possibility that 1 of them, especially the exon IX polymorphism, is associated with subtypes of myocardial infarction or to evolutive particularities that were not assessed in this study, cannot be excluded and is currently being evaluated.

Spectrin deficiency is the most common deficiency found in HS. It is heterogeneous in terms of clinical expression, inheritance (dominant or recessive) and underlying genetic defects (related to alpha- or beta-spectrin gene defects or secondary to ankyrin gene defects). We studied a sampling of French dominant HS families, selected after linkage analyses, and found the presence of mutations resulting in the silencing of the mutant beta-spectrin allele. In three HS families, one haploid set of beta-spectrin mRNA was undectectable. In two families, a deletion of 8 bases (leading to a frameshift and a premature stop codon) and a nonsense mutation were identified, respectively. In the third HS family, we were unable to characterize a relevant mutation but the loss of heterozygosity at the cDNA level suggested the presence of a null mutation of the beta-spectrin allele. Sequencing of the beta-spectrin gene has also uncovered several new polymorphisms in the coding region of the beta-spectrin gene which will be very useful for detecting loss of heterozygosity at the cDNA level and designating the beta-spectrin gene as the culprit one.

We studied a family with autosomal dominant hereditary spherocytosis (HS) associated with a mild spectrin deficiency. Linkage analysis using two microsatellite markers (D14S63 and D14S271) very close to the β‐spectrin gene (SPTB) showed that HS co‐segregated with alleles of these microsatellite markers and the linkage between the marker and HS was statistically significant. The presence of a β‐spectrin protein polymorphism (β‐spectrin Vay; A1880V) in trans of the HS allele was not itself deleterious, but allowed the detection of decreased membrane expression of the spherocytic β‐spectrin allele in two HS‐affected subjects. Direct sequencing of the coding exons of the β‐spectrin gene in one affected subject showed the presence of a G → C transversion at the terminal nucleotide of exon 3, which did not change the leucine codon 100 (CTG → CTC). The presence of the mutation was confirmed by restriction enzyme digestion at the DNA level in all affected SH members of the family. The G → C mutation severely reduced the utilization of the 5′ splice site and resulted in aberrant mRNA splicing with intron 3 retention.

Among 80 hereditary spherocytosis (HS) kindreds studied using denaturing electrophoretic separation of solubilized eythrocyte membrane proteins, we recognized three prominent subsets: HS with isolated spectrin deficiency, HS with combined spectrin and ankyrin deficiency, and HS with band 3 deficiency. These three subsets represent more than 80% of the HS kindreds studied. In this study, eight dominant HS kindreds with band 3 deficiency were investigated for band 3 mutations. In three of these kindreds, linkage analyses confirmed the band 3 gene as the culprit gene. In an attempt to identify the responsible mutations, denaturing gradient gel electrophoresis (DGGE) was used to explore the coding exons (exons 2–20) of band 3 gene. Five different mutations were found in the eight kindreds. In five kindreds we identified substitutions of highly conserved residues, positioned at boundaries of putative transmembrane segments: a C → T substitution at codon 490 changed arginine (CGC) to cysteine (TGC) in three kindreds, a C → T substitution at codon 837 changed threonine (ACG) to methionine (ATG) in two kindreds. In the sixth kindred a G deletion was found in a stretch of five G starting at position 1475, leading to a stop codon either at position 1527 or 1565. In the seventh kindred a T deletion at position 1600 resulted in a stop codon at position 1733 and in the last kindred a T deletion was identified at position 355, leading to a stop codon at position 447. The mutant transcript was present in HS patients bearing missense mutations, whereas only the normal transcript was found in HS patients with frameshift mutations. In the latter group the mean decrease in membrane band 3 content was significantly lower, leading to speculation that missense mutations may have some sort of dominant negative effect.

Hereditary spherocytosis (HS) is an inherited hemolytic anemia characterized by the presence of dense spherocytic red cells. In HS patients, red cell membrane protein gel electrophoresis has identified different subsets of abnormalities: isolated spectrin deficiency, combined spectrin and ankyrin deficiency, band 3 deficiency. To direct the search for the molecular defect in 9 families with dominant HS, we developed microsatellite markers specific for the membrane protein encoding genes possibly involved in HS (alpha- and beta-spectrin, ankyrin and band 3 genes) and genotyped each family. In 5 families with isolated spectrin deficiency, the beta-spectrin gene was designated as candidate. In one family with combined spectrin/ankyrin deficiency, only the ankyrin gene was not excluded, whereas in the 3 HS families with band 3 deficiency, only the band 3 gene was not excluded. This work allowed development of a reliable methodology to search for candidate genes in HS and showed the frequent involvement of the beta-spectrin gene in HS with isolated spectrin deficiency.

We studied an African population in Benin and discovered an unexpectedly high frequency (1.6%) of hereditary elliptocytosis (HE) among the 1447 subjects studied. In approximately two‐thirds of HE individuals we identified molecular defects, primarily those in erythrocyte α‐spectrin (dupL154, L260P and L207P mutations), as well as a novel mutation of erythrocyte β‐spectrin (β‐W2061R mutation). We also identified the genetic basis of a previously identified protein polymorphism of the αIII domain of spectrin (R1331I mutation). The genetic background of HE in the African population was studied using a number of polymorphisms of the α‐spectrin gene, including the αIII domain polymorphism. These studies suggest that the HE mutations appear to have originated from separate genetic backgrounds in this population.

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

Human MutationVolume 5, Issue 4 p. 339-340 Mutation in Brief Protein 4.1 Lille, a novel mutation in the downstream initiation codon of protein 4.1 gene associated with heterozygous 4,1(−) hereditary elliptocytosis M. Garbarz, Corresponding Author M. Garbarz Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorI. Devaux, I. Devaux Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorO. Bournier, O. Bournier Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorB. Grandchamp, B. Grandchamp Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorD. Dhermy, D. Dhermy Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this author M. Garbarz, Corresponding Author M. Garbarz Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorI. Devaux, I. Devaux Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorO. Bournier, O. Bournier Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorB. Grandchamp, B. Grandchamp Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this authorD. Dhermy, D. Dhermy Génétique et Pathologie Moléculaires de L'Hématopoïése, Inserum U. 409, Faculté de Médecine X. Bichat, B.P. 416, 75870 Paris Cedex 18, France; Fax: 33-1-42264 624Search for more papers by this author First published: 1995 https://doi.org/10.1002/humu.1380050412Citations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References Alloisio N, Morlé L, Dorléac E, Gentilhomme O, Bachir D, Guetarni D, Colonna P, Bost M, Zouaoui Z, Roda L, Roussel D, Delaunary J (1985) The heterozygous form of the 4.1(—) hereditary elliptocytosis [the 4.l(—)trait]. Blood 65: 46– 51. Conboy JG (1993) Structure, function, and molecular genetics of erythroid membrane skeletal protein 4.1 in normal and abnormal red blood cells. Semin Hematol 30: 58– 73. Dalla Venezia N, Gilsanz F, Alloisio N, Ducluzeau MT, Benz EJ, Jr, Delaunay J (1992) Homozygous 4.1(—) heterditary elliptocytosis associated with a point mutation in the downstream initiation codon of protein 4.1 gene. J Clin Invest 90: 1713– 1717. Feddal S, Brunet G, Roda L, Chabanis S, Alloisio N, Morlé L, Ducluzeau MT, Maréchal J, Robert JM, Benz EJ, Delaunary J, Baklouti F (1991) Molecular analysis of hereditary elliptocytosis with reduced protein 4.1 in the French northern Alps. Blood 78: 2113– 2119. Palek J, Jarolim P (1993) Clinical expression and laboratory detection of red blood cell membrane protein mutations. Semin Hematol 30: 249– 283. Parquet N, Devaux I, Boulanger L, Galand C, Boivin P, Lecomte MC, Dhermy D, Garbarz M (1994) Identification of three novel spectrin αI/74 mutations in hereditary elliptocytosis. Further support for a triple-stranded folding unit model of the spectrin heterodimer contact site. Blood 84: 303– 308. Saiki RK, Gelfand DH, Stoffel S, Sharf S, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487– 491. Citing Literature Volume5, Issue41995Pages 339-340 ReferencesRelatedInformation

Allele alpha LELY is a low-expression allele of erythroid spectrin alpha-chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele alpha LELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious alpha-alleles occurring in trans, and as such has clinical importance. The aim of this study was to evaluate the incidence of allele alpha LELY in various ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele alpha LELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low-expression feature, whenever they could be assessed, were invariably observed. Allele alpha LELY appears to be an ancient and stable allele.

Six individuals with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) from three unrelated families were evaluated. Defects in the ability of spectrin (Sp) to undergo self-association were present, and associated with increased recovery of the Sp alpha I 74-kD fragment after limited tryptic digestion (Sp alpha I/74 variant). Because mutations associated with the Sp alpha I/74 variant described to date have been localized to the 5′ coding region of the alpha-Sp gene (exon 2) or at the 3′ coding end of the beta-Sp gene (exon 30), the polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) method was used to detect mutations in these two regions. In one family with HE, an abnormal pattern of migration of PCR- amplified fragments containing exon 2 was observed, and led to the detection of a new mutation (Ile24Ser) in helix 3 of repeating segment alpha 1. In the two other families, an abnormal pattern of migration of PCR-amplified fragments containing exon 30 was observed in affected individuals, and sequencing led to the identification of two new mutations (Ala2023Val and Trp2024Arg) in helix 1 of repeating segment beta 17. The elliptogenic potential of these mutations emphasizes the importance of the conformational integrity of each of the three helices involved in the formation of the Sp heterodimer contact site, and will help identify critical amino acids involved in this interaction.

Summary. Allele αLELY is a low‐expression allele of the erythroid spectrin α‐gene. It carries mutations in exon 40 (αV/41 polymorphism) and intron 45, respectively, and is associated with partial skipping of exon 46. The latter phenomenon is thought to impair the recruitment of α‐chains by β‐chains, and would eventually account for the low‐expression character. When it occurs in trans to an α‐allele responsible for hereditary elliptocytosis (αHE allele; αHE/αLELY diplotype), allele αLELY enhances the severity of elliptocytosis. Because allele αLELY is widespread, we anticipated that it would occasionally carry HE determinants. These variants of allele αLELY will be designated αHE‐LELY alleles. We report two families with the same αHE‐LELY allele. The HE component was the known α28 Arg → His mutation. This αHE‐LELY allele was investigated within the αHE‐LELY/αLELY diplotype, a diplotye not described before. Except for the neonatal period, the presentation was mild. In a consistent manner, the αLELY component in cis of the αHE mutation counteracted the like component in trans.