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Heterogenous band 3 deficiency in hereditary spherocytosis related to different band 3 gene defects
D Dhermy1, C Galand, O Bournier
1INSERM U409, Centre Claude Bernard, Faculté X. Bichat, Paris, France.
Insights
Hereditary spherocytosis (HS) is often caused by band 3 gene mutations. This study identified five novel band 3 mutations in HS patients, revealing potential dominant negative effects in missense mutations.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Hereditary spherocytosis (HS) is a genetic blood disorder characterized by spherical red blood cells.
- Three main HS subsets exist: isolated spectrin deficiency, combined spectrin and ankyrin deficiency, and band 3 deficiency, accounting for over 80% of cases.
Purpose of the Study:
- To investigate band 3 gene mutations in eight dominant hereditary spherocytosis kindreds with band 3 deficiency.
- To identify specific mutations within the band 3 gene responsible for HS.
Main Methods:
- Linkage analysis to confirm the band 3 gene as the causative gene.
- Denaturing gradient gel electrophoresis (DGGE) to screen coding exons (2-20) of the band 3 gene for mutations.
- Analysis of mutant transcript presence and band 3 content in patients.
Main Results:
- Three of eight kindreds showed linkage to the band 3 gene.
- Five distinct band 3 gene mutations were identified across the eight kindreds.
- Missense mutations (codon 490, 837) and frameshift mutations (deletions at positions 1475, 1600, 355) were found.
- Mutant transcripts were detected in missense mutation cases, while only normal transcripts were found in frameshift mutation cases.
Conclusions:
- Band 3 gene mutations are a significant cause of hereditary spherocytosis.
- Missense band 3 mutations may exert a dominant negative effect, leading to reduced band 3 content.
- Frameshift band 3 mutations result in lower reductions in band 3 content compared to missense mutations.
Abstract:
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.