Clinical and Haematologic Features of 300 Patients Affected by Hereditary Spherocytosis as a Function of the Type of the Membrane Protein Defect.

Hereditary Spherocytosis (HS) is caused by defects of red cell membrane proteins (spectrin, ankyrin, band 3 and band 4.2). The aim of this study is to analyse a large database of 300 HS patients grouped according to SDS-PAGE, 1) to ascertain whether the clinical/haematological features and response to splenectomy are related to the type of molecular defect, and 2) to compare the sensitivity of the most common laboratory screening tests for HS in the various subsets of patients. Three hundred consecutive patients were investigated; 41 patients had been splenectomised before the time of the study, and 21 thereafter. In not splenectomised subjects, anaemia was severe in 7% of cases, mild to moderate in 52% and compensated in the remaining cases. The median number of spherocytes at the peripheral blood smear examination was 7%. The most frequent protein abnormalities revealed by SDS-PAGE were band 3 (54%) and spectrin and/or ankyrin (34%) deficiency. The membrane protein defect was undetectable in 3% of splenectomised versus 11% of not splenectomised patients. In patients evaluated by SDS-PAGE before and after splenectomy surgery allowed the identification of the defect (one band 3 and seven spectrin/ankyrin deficiency) in all the 8 previously unclassified cases. No significant differences were observed among clinical and haematological parameters of patients grouped according to the type of biochemical defect, although the degree of anaemia, haemolysis markers and median spherocyte number were higher in spectrin deficient than in the other groups of patients. Splenectomy was clinically effective in correcting both anaemia and haemolysis, but splenectomised spectrin/ankyrin deficient patients showed after splenectomy a slightly lower median rise in haemoglobin, and a higher reticulocyte number and unconjugated bilirubin level than band 3 deficient patients. The red cell osmotic fragility tests’ sensitivity varies greatly and ranged from 48 to 95%. The sensitivity was similar in patients with band 3 and spectrin/ankyrin deficiency and also in patients without detectable membrane defect. Furthermore, the sensitivity of all the methods investigated increased in splenectomised cases. AGLT displayed the highest sensitivity, and the association of AGLT and NaCl test on incubated blood reached a sensitivity of 99%, enabling the diagnosis of the atypical HS cases, such as those with rare or no spherocytes in blood smears, normal MCHC and reticulocyte counts.

Comparison of the ankyrin (AC)n microsatellites in genomic DNA and mRNA reveals absence of one ankyrin mRNA allele in 20% of patients with hereditary spherocytosis

Combined deficiency of ankyrin and spectrin represents the most common biochemical abnormality in hereditary spherocytosis (HS). To examine whether a decrease in ankyrin mRNA represents a frequent cause of this type of HS, we took advantage of the reported (AC)n microsatellite polymorphism in the 3′ untranslated region of ankyrin cDNA. We first measured the number of AC repeats in genomic DNA encoding erythrocyte ankyrin in 36 unrelated Czech HS patients with combined ankyrin and spectrin deficiency and found 21 of these subjects (58%) to be heterozygotes for the (AC)n microsatellite size. Further analysis of reticulocyte RNA showed that ankyrin cDNA from 7 of these 21 heterozygotes (33%) contained only one of the two ankyrin alleles. We conclude that approximately 1/3 of ankyrin-deficient autosomal dominant HS is caused by reduced expression of one ankyrin allele which, in turn, is caused by either a reduced transcription of one allele of the mutated ankyrin gene or abnormal processing or decreased stability of the mutant ankyrin mRNA. Because ankyrin deficiency is detected in approximately 60% of HS subjects, this result suggests that approximately 20% of all HS is caused by a decreased expression of one ankyrin mRNA allele.

Combined spectrin and ankyrin deficiency is common in autosomal dominant hereditary spherocytosis

The common autosomal dominant form of hereditary spherocytosis (HS) has been genetically linked to defects of the erythroid ankyrin gene in a few families; however, the frequency of ankyrin deficiency and its relationship to red blood cell (RBC) spectrin content are unknown. To test these questions, we measured RBC spectrin and ankyrin by radioimmunoassay in 39 patients from 20 families with dominant HS. Normal RBCs contained 242,000 +/- 20,500 spectrin heterodimers and 124,500 +/- 11,000 ankyrins per cell. In dominant HS, RBC spectrin and ankyrin ranged from about 40% to 100% of normal and were continuously distributed. Measurements in the same patient on different occasions were reproducible (+/- 5% to 10%) and RBCs from affected members of a kindred contained similar amounts of spectrin and ankyrin (+/- 3% to 4%). Spectrin and ankyrin levels were almost always less than the assay controls, but were less than the normal range in only 75% and 80% of kindreds, respectively. Remarkably, the degree of RBC spectrin and ankyrin deficiency was very similar in 19 of 20 HS kindreds. One otherwise typical family differed, with marked ankyrin deficiency (45% of control) and a relatively mild spectrin deficit (81%). We conclude that most patients with dominant HS have combined ankyrin and spectrin deficiency and that the two proteins are usually about equally deficient, suggesting that defects in ankyrin expression, ankyrin stability, or ankyrin band 3 (AE1) interactions may be common in dominant HS.

Combined spectrin and ankyrin deficiency is common in autosomal dominant hereditary spherocytosis

The common autosomal dominant form of hereditary spherocytosis (HS) has been genetically linked to defects of the erythroid ankyrin gene in a few families; however, the frequency of ankyrin deficiency and its relationship to red blood cell (RBC) spectrin content are unknown. To test these questions, we measured RBC spectrin and ankyrin by radioimmunoassay in 39 patients from 20 families with dominant HS. Normal RBCs contained 242,000 +/- 20,500 spectrin heterodimers and 124,500 +/- 11,000 ankyrins per cell. In dominant HS, RBC spectrin and ankyrin ranged from about 40% to 100% of normal and were continuously distributed. Measurements in the same patient on different occasions were reproducible (+/- 5% to 10%) and RBCs from affected members of a kindred contained similar amounts of spectrin and ankyrin (+/- 3% to 4%). Spectrin and ankyrin levels were almost always less than the assay controls, but were less than the normal range in only 75% and 80% of kindreds, respectively. Remarkably, the degree of RBC spectrin and ankyrin deficiency was very similar in 19 of 20 HS kindreds. One otherwise typical family differed, with marked ankyrin deficiency (45% of control) and a relatively mild spectrin deficit (81%). We conclude that most patients with dominant HS have combined ankyrin and spectrin deficiency and that the two proteins are usually about equally deficient, suggesting that defects in ankyrin expression, ankyrin stability, or ankyrin band 3 (AE1) interactions may be common in dominant HS.