Low expression allele alpha LELY of red cell spectrin is associated with mutations in exon 40 (alpha V/41 polymorphism) and intron 45 and with partial skipping of exon 46.

The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).

Homozygous 4.1(-) hereditary elliptocytosis associated with a point mutation in the downstream initiation codon of protein 4.1 gene.

We studied a 43 yr-old Spanish patient with homozygous 4.1(-) hereditary elliptocytosis. Any form of protein 4.1 was missing in the red cells. Spectrin and actin were slightly, yet significantly, diminished. Alterations appeared at the level of proteins 4.5 and 4.9. Glycophorin C was sharply reduced. The abnormal allele was associated with the -++-- haplotype (Pvu II, Bgl II, Bgl II, Pvu II, Pvu II). mRNA 4.1(-) had an apparently normal size but was diminished by about two-thirds. Because the abnormal phenotype pertained to the red cell, we sequenced the 4.1 cDNA regions that appear critical to this cell type. The ultimate change turned out to be a point mutation of the downstream translation initiation codon (AUG-->AGG). No disorders in other cell types could be related with certainty to the present 4.1(-) HE allele.

Sp alpha V/41: a common spectrin polymorphism at the alpha IV-alpha V domain junction. Relevance to the expression level of hereditary elliptocytosis due to alpha-spectrin variants located in trans.

Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.

Protein 4.1 deficiency associated with an altered binding to the spectrin-actin complex of the red cell membrane skeleton.

Protein 4.1 has been defined as a major component of the subcortical skeleton of erythrocytes. It binds the spectrin--actin scaffold through a 10-kD internal domain. This binding requires an essential 21-amino acid sequence motif, Motif I, which is retained by alternative splicing at the late stage of erythroid differentiation. We here analyze the molecular basis of heterozygous 4.1(-) hereditary elliptocytosis, associated with protein 4.1 partial deficiency, in nine related French families. cDNA sequencing revealed a single codon deletion (AAA) resulting in a lysine residue deletion within the 10-kD binding domain, 3' of Motif I. The mutated allele was designated allele 4.1 Aravis. In order to assess the functional effect of the codon deletion, recombinant 10-kD constructs were made and various binding assays were performed using spectrin, purified spectrin-actin complex, or red cell membranes. These experiments demonstrated that the deletion of the Lys residue clearly prevents the binding capacity. Similar results were obtained with a construct containing the Lys residue but lacking Motif I. These data strongly suggest that the binding site to the spectrin-actin complex must contain the Lys 447 (or 448), and therefore resides not only on Motif I but extends 3' of this essential motif.

Spectrin beta-chain variant associated with hereditary elliptocytosis.

An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase. The presence of the beta'-chain was associated with a defective self-association of spectrin dimer to form tetramer as manifested by: (a) an excess of spectrin dimer in the 4 degrees C spectrin crude extract, (b) a defective self-association of the spectrin dimer in the 37 degrees C crude spectrin extracts. Gel electrophoretic analysis of the tetramer and dimer species isolated from the proband's 4 degrees C extract showed that the tetramer contained trace amounts of the beta'-chain, whereas in contrast, a large proportion of beta'-chain was present in the dimer. These results demonstrated the responsibility of the beta'-chain for the defective reassociation of spectrin dimer into tetramer. The study of this abnormal spectrin confirms the participation of spectrin beta-chain in dimer-dimer association and strongly suggests that the phosphorylation sites of the normal beta-chain are located at the end of the molecule involved in the dimer-dimer interactions.

Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe).

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.

Truncated hemoglobins in actinorhizal nodules of Datisca glomerata.

Three types of hemoglobins exist in higher plants, symbiotic, non-symbiotic, and truncated hemoglobins. Symbiotic (class II) hemoglobins play a role in oxygen supply to intracellular nitrogen-fixing symbionts in legume root nodules, and in one case ( Parasponia Sp.), a non-symbiotic (class I) hemoglobin has been recruited for this function. Here we report the induction of a host gene, dgtrHB1, encoding a truncated hemoglobin in Frankia-induced nodules of the actinorhizal plant Datisca glomerata. Induction takes place specifically in cells infected by the microsymbiont, prior to the onset of bacterial nitrogen fixation. A bacterial gene (Frankia trHBO) encoding a truncated hemoglobin with O (2)-binding kinetics suitable for the facilitation of O (2) diffusion ( ) is also expressed in symbiosis. Nodule oximetry confirms the presence of a molecule that binds oxygen reversibly in D. glomerata nodules, but indicates a low overall hemoglobin concentration suggesting a local function. Frankia trHbO is likely to be responsible for this activity. The function of the D. glomerata truncated hemoglobin is unknown; a possible role in nitric oxide detoxification is suggested.

Red cell membrane sialoglycoprotein beta in homozygous and heterozygous 4.1(-) hereditary elliptocytosis.

Sialoglycoprotein beta, a minor sialoglycoprotein of the red cell membrane, was studied in homozygous and heterozygous 4.1(-) hereditary elliptocytosis, a variety of hereditary elliptocytosis characterized by total or partial absence of protein 4.1. Erythrocytes were treated with the periodic acid-NaB3H4 procedure. Following polyacrylamide gel electrophoresis in the presence of SDS, labelled sialoglycoproteins were revealed by fluorography. (i) In the ghosts from the 4.1(-) homozygote, sialoglycoprotein beta was sharply decreased. It is not sure whether the residual material is sialoglycoprotein beta itself, or a distinct sialoglycoprotein migrating in the same place. In long exposure fluorograms, sialoglycoprotein gamma (a sialoglycoprotein related to sialoglycoprotein beta) also turned out to be reduced. In the homozygote's Triton-shells, sialoglycoprotein beta and gamma appeared completely absent. (ii) In the 4.1(-) heterozygote, sialoglycoprotein beta appeared slightly reduced, whereas sialoglycoprotein gamma appeared normal. Both of these proteins were extracted in seemingly normal amounts in the Triton-shells. These observations bring further support to the view that there is an interaction between skeletal membrane protein 4.1 and sialoglycoprotein beta, that is additional to other interactions between the former protein and the lipid bilayer and/or other transmembrane proteins.

A novel C202F mutation in the connexin26 gene (GJB2) associated with autosomal dominant isolated hearing loss.

Mutations in the GJB2 gene encoding connexin26 (CX26) account for up to 50% of cases of autosomal recessive hearing loss. In contrast, only one GJB2 mutation has been reported to date in an autosomal dominant form of isolated prelingual hearing loss. We report here a novel heterozygous 605G-->T mutation in GJB2 in all affected members of a large family with late childhood onset of autosomal dominant isolated hearing loss. The resulting C202F substitution, which lies in the fourth (M4) transmembrane domain of CX26, may impair connexin oligomerisation. Finally, our study suggests that GJB2 should be screened for heterozygous mutations in patients with autosomal dominant isolated hearing impairment, whatever the severity of the disease.

Mutation in the zonadhesin-like domain of alpha-tectorin associated with autosomal dominant non-syndromic hearing loss.

A gene responsible for autosomal dominant non-syndromic hearing impairment in two families (DFNA8 and DFNA12) has recently been identified as TECTA encoding alpha-tectorin, a major component of the tectorial membrane. In these families, missense mutations within the zona pellucida domain of alpha-tectorin were associated with stable severe mid-frequency hearing loss. The present study reports linkage to DFNA12 in a new family with autosomal dominant high frequency hearing loss progressing from mild to moderate severity. The candidate region refined to 3.8 cM still contained the TECTA gene. A missense mutation (C1619S) was identified in the zonadhesin-like domain. This mutation abolishes the first of the vicinal cysteines (1619Cys-Gly-Leu- 1622Cys) present in the D4 von Willebrand factor (vWf) type D repeat. These results further support the involvement of TECTA mutations in autosomal dominant hearing impairment, and suggest that vicinal cysteines are involved in tectorial membrane matrix assembly.

[Genetic deafness:the primary cause of sensorineural hearing loss in children]. / Les surdités génétiques: première cause de surdité de perception de l'enfant.

Genetically-transferred hearing impairments account for more than 50% of cases of pediatric sensorineural hearing defects. Multiple clinical aspects are involved in genetic hearing impairment, including the involvement of other organs, genetic inheritance, and the degree and age at onset of hearing loss. Diagnosis relies on family history, on the systematic investigation of the symptomatology including an associated syndrome, and audiometry testing in parents and siblings. Analysis of the connexin 26 gene is also indicated, as it is frequently involved in this disorder. Further genetic analysis in affected families will aid in detecting other as yet unidentified genes responsible for hearing impairment.

The genetic disorders of the red cell skeleton.

The genetic disorders of the red cell skeleton encompass hereditary spherocytosis, hereditary elliptocytosis and an array of ill-defined haemolytic anaemias. Protein chemistry and molecular genetics have illuminated the supramolecular arrangement of the skeleton, the sequence and three-dimensional structure of its protein components, the exon-intron organization of the corresponding genes, the complex splicing undergone by their transcripts. Basically, hereditary spherocytosis is often due to a defect of ankyrin, hereditary elliptocytosis usually results from alterations of spectrin or protein 4.1. Other conditions are related to changes in the anion transporter or protein 4.2. The heterogeneity of the genomic changes, their ultimate consequences at the protein level open windows on fundamental problems concerning alternative splicing of mRNAs and structure-function relationships in proteins.

Spectrin mutations in hereditary elliptocytosis and hereditary spherocytosis.

Hereditary elliptocytosis (HE), its aggravated form hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS) designate a set of congenital hemolytic syndromes. The responsible mutations lie in several genes encoding proteins of the red cell membrane. In particular, they involve the SPTA1 and SPTB genes that encode erythroid spectrin alpha- and beta-chains, respectively. In situ, spectrin is a alpha 2 beta 2 fibrillar tetramer resulting from the head-to-head self-association of two alpha beta dimers. In HE, the 24 known alpha-chain mutations lie in the self-association site or its vicinity, whereas the 17 beta-chain mutations occur in the self-association site itself (record of November 30, 1995). Allele alpha LELY (LELY: Low Expression LYon) is found in ethnic groups remote from one another with a uniform frequency (20-30% of all alpha-alleles). It allows an expanded expression of any HE alpha-allele located in trans and results in severe HE or in HPP. In HS, a number of spectrin mutations have been recorded recently. Allele alpha LEPRA (LEPRA: Low Expression PRAgue) would occur in a recurrent fashion.

Analysis of the red cell membrane in a family with hereditary elliptocytosis--total or partial of protein 4.1.

In a 12-year-old boy carrying a clinically silent elliptocytosis, we observed a total lack of red cell membrane band 4.1. Band 4.1 was partially absent in the father who also displayed a clinically silent elliptocytosis and, remarkably, in the mother although she presented normal discocytes. Band (2 and 2.1.) phosphorylation was sharply reduced in the three persons examined. In the propositus and his mother, but not in his father, a clearly phosphorylated band appeared at the level of band 4.2. We suggest that the father and the mother carry two distinct alleles affecting differently the interactions within the spectrin-actin protein 4.1 complex. The father's allele is elliptocytogenic in the heterozygous state and, among other molecular alterations, prevents the attachment of protein 4.1. The mother's allele is morphologically silent in the heterozygous state, yet it also affects the binding of protein 4.1, possibly because the latter is shortened. The propositus, being doubly heterozygous, has the same morphological phenotype as his father, but his protein 4.1 electrophoretic phenotype is the addition of both parental phenotypes. The distinct phosphorylation patterns in the region of bands 4.1 and 4.2 are also consistent with the two-allele hypothesis.

The genetic abnormalities involving red cell membrane protein 4.1 with or without elliptocytosis.

We have studied 20 caucasian individuals including 3 independent persons and 17 persons belonging to 6 unrelated families. These subjects are normal or elliptocytic. On biochemical grounds, families with hereditary elliptocytosis (HE) can be divided in two groups. (i) Three families in which HE is associated with a marked reduction (approximately 30%) of the band 4.1 percentage, upon scanning of SDS-polyacrylamide gels. This deficiency is associated with the absence of obvious clinical symptom and a dominant genetic transmission. (ii) Three HE families display a normal amount of band 4.1. Among two of them, HE is concomitant with moderate clinical manifestations and with a genetic transmission that seems to be morphologically recessive. In the last family, HE is transmitted as a dominant trait and is clinically silent. Finally, in the 3 isolated persons, HE is asymptomatic but the mode of genetic transmission could not be ascertained. One of them has a decreased amount of band 4.1 similar to that observed in the first group. To date, the band 4.1 deficiency is probably the best example of a relatively frequent specific molecular defect associated with a specific morphological abnormality. In one family with this type of HE, we have fortuitously discovered, at the heterozygous state, a variant of protein 4.1, shortened by about 8 500 daltons, involving both subcomponents 4.1a and 4.1b and morphologically silent.