Elisa Fermo

Red cell pyruvate kinase deficiency: molecular and clinical aspects

Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of the glycolytic pathway causing hereditary non‐spherocytic haemolytic anaemia. The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life‐threatening neonatal anaemia and jaundice necessitating exchange transfusions. Erythrocyte PK is synthesized under the control of the PK‐LR gene located on chromosome 1. To date, more than 150 different mutations in the PK‐LR gene have been associated with PK deficiency. First attempts to delineate the biochemical and clinical consequences of the molecular defect were mainly based on the observation of the few homozygous patients and on the analysis of the three‐dimensional structure of the enzyme. More recently, the comparison of the recombinant mutants of human red cell PK with the wild‐type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined and help to correlate genotype to clinical phenotype.

Congenital dyserythropoietic anemia type II (CDAII) is caused by mutations in the SEC23B gene

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease characterized by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypoglycosylation of some red blood cell (RBC) membrane proteins. Recent studies indicated that CDAII is caused by a defect disturbing Golgi processing in erythroblasts. A linkage analysis located a candidate region on chromosome 20, termed the CDAN2 locus, in the majority of CDAII patients but the aberrant gene has not so far been elucidated. We used a proteomic‐genomic approach to identify SEC23B as the candidate gene for CDAII by matching the recently published data on the cytoplasmic proteome of human RBCs with the chromosomic localization of CDAN2 locus. Sequencing analysis of SEC23B gene in 13 CDAII patients from 10 families revealed 12 different mutations: six missense (c.40C>T, c.325G>A, c.1043A>C, c.1489C>T, c.1808C>T, and c.2101C>T), two frameshift (c.428_428delAinsCG and c.1821delT), one splicing (c.689+1G>A), and three nonsense (c.568C>T, c.649C>T, and c.1660C>T). Mutations c.40C>T and c.325G>A were detected in unrelated patients. SEC23B is a member of the Sec23/Sec24 family, a component of the COPII coat protein complex involved in protein transport through membrane vesicles. Abnormalities in this gene are likely to disturb endoplasmic reticulum (ER)‐to‐Golgi trafficking, affecting different glycosylation pathways and ultimately accounting for the cellular phenotype observed in CDAII. Hum Mutat 30:1–7, 2009.

Clinical and hematologic features of 300 patients affected by hereditary spherocytosis grouped according to the type of the membrane protein defect

The molecular basis of hereditary spherocytosis is highly heterogeneous, involving the genes encoding for spectrin, ankyrin, band 3 and protein 4.2. The findings of this retrospective study show that splenectomy corrected anemia in patients with all molecular subtypes of hereditary spherocytosis. Thus, the definition of the red cell membrane defect in hereditary spherocytosis has no major clinical implications, but may be useful for a differential diagnosis from other hematologic disorders that mimic this hemolytic anemia. See related perspective article on page 1283. Background Hereditary spherocytosis is a very heterogeneous form of hemolytic anemia. The aim of this study was to relate the type of molecular defect with clinical and hematologic features and response to splenectomy using information from a large database of patients. Design and Methods Data from 300 consecutive patients with hereditary spherocytosis, grouped according to the results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, were analyzed and the sensitivity of red cell osmotic fragility tests was compared in various subsets of patients. Results Band 3 and spectrin deficiencies were the most common protein abnormalities (54% and 31%, respectively); 11% of cases were not classified by the electrophoretic analysis. Spectrin deficiency was more frequently diagnosed in childhood and band 3 deficiency in adulthood. Hemoglobin concentration was slightly lower, spherocyte number and hemolysis markers higher in spectrin deficiency than in band 3 deficiency. The sensitivity of the osmotic fragility tests ranged from 48% to 95%, and was independent of the type and amount of the membrane defect. The association of the acidified glycerol lysis test and the NaCl test on incubated blood reached a sensitivity of 99%. Splenectomy corrected the anemia in patients with all subtypes of hereditary spherocytosis although spectrin-deficient patients still showed increased reticulocyte numbers and levels of unconjugated bilirubin. Splenectomy allowed the identification of the membrane defect in all the previously unclassified patients, most of whom had spectrin and/or ankyrin deficiency. Conclusions The definition of the red cell membrane defect in hereditary spherocytosis has no major clinical implications, but may be useful for a differential diagnosis from other hematologic disorders that mimic this hemolytic anemia.

Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics

Background The laboratory diagnosis of hereditary spherocytosis commonly relies on NaCl-based or glycerol-based red cell osmotic fragility tests; more recently, an assay directly targeting the hereditary spherocytosis molecular defect (eosin-5′-maleimide-binding test) has been proposed. None of the available tests identifies all cases of hereditary spherocytosis. Design and Methods We compared the performances of the eosin-5′-maleimide-binding test, NaCl-osmotic fragility studies on fresh and incubated blood, the glycerol lysis test, the acidified glycerol lysis test, and the Pink test on a series of 150 patients with hereditary spherocytosis grouped according to clinical phenotype and the defective protein, with the final aim of finding the combination of tests associated with the highest diagnostic power, even in the mildest cases of hereditary spherocytosis. Results The eosin-5′-maleimide-binding test had a sensitivity of 93% and a specificity of 98% for detecting hereditary spherocytosis: the sensitivity was independent of the type and amount of molecular defect and of the clinical phenotype. The acidified glycerol lysis test and Pink test showed comparable sensitivity (95% and 91%). The sensitivity of NaCl osmotic fragility tests, commonly considered the gold standard for the diagnosis of hereditary spherocytosis, was 68% on fresh blood and 81% on incubated blood, and further decreased in compensated cases (53% and 64%, respectively). The combination of the eosin-5′-maleimide-binding test and acidified glycerol lysis test enabled all patients with hereditary spherocytosis to be identified. The eosin-5′-maleimide-binding test showed the greatest disease specificity. Conclusions Each type of test fails to diagnose some cases of hereditary spherocytosis. The association of an eosin-5′-maleimide-binding test and an acidified glycerol lysis test enabled identification of all patients with hereditary spherocytosis in this series and, therefore, represents a currently effective diagnostic strategy for hereditary spherocytosis including mild/compensated cases.

Molecular insights on pathogenic effects of mutations causing phosphoglycerate kinase deficiency.

Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.

Hereditary pyrimidine 5'-nucleotidase deficiency: from genetics to clinical manifestations.

Hereditary pyrimidine 5′‐nucleotidase (P5′N) deficiency is the most frequent abnormality of the red cell nucleotide metabolism causing hereditary non‐spherocytic haemolytic anaemia. The disorder is usually characterised by mild‐to‐moderate haemolytic anaemia associated with the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. The precise mechanisms leading to the destruction of P5′N deficient red cells are still unclear. The pyrimidine 5′‐nucleotidase type‐I (P5′N‐1) gene is localised on 7p15‐p14 and the cDNA has been cloned and sequenced; 20 mutations have been identified so far in 30 unrelated families, most of them at the homozygous level. Recently, the comparison of recombinant mutants of human P5′N‐1 with the wild‐type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined and help to correlate genotype to clinical phenotype.

Red cell pyruvate kinase deficiency: 17 new mutations of the PK-LR gene

The PK‐LR gene was studied in 23 patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase (PK) deficiency. Twenty‐seven different mutations were detected among the 42 mutated alleles identified: 19 missense mutations, four splice site mutations and one nonsense, one single base deletion and two large deletions. Seventeen of them (107G, 278T, 403T, 409A, 661A, 859C, 958A, 1094T, 1190T, 1209A, 1232C, 1369G, 507A, IVS9 −1c, IVS9 +43t, del C224, del 5006bp IVS3→ nt 1431) were new. Although all the exons, the flanking regions and the promoter were sequenced in all cases, we failed to detect the second expected mutation in four subjects. To correlate genotype to phenotype, the molecular results were related to the biochemical properties of the mutant enzymes by an analysis of the three‐dimensional structure of erythrocyte PK. The new mutant 409A, found in association with the large deletion of 5006 bp in a newborn baby who died soon after birth, was functionally characterized by mutagenesis and in vitro expression of the protein to investigate its contribution in the severity of the clinical pattern. However, the biochemical data obtained for the mutant enzyme cannot explain the severe anaemia found in the PK‐deficient patient hemizygous for this mutation.

Hereditary red cell membrane defects: diagnostic and clinical aspects

The plasma membrane of the erythrocyte accounts for all of this cell’s antigenic, transport, and mechanical characteristics, particularly its ability to undergo large passive deformations during repeated passage through the narrow capillaries of the microvasculature, throughout its 120-day life span. The determinant of normal membrane cohesion is the system of “vertical” linkages between the phospholipid bilayer and membrane skeleton, formed by the interactions of the cytoplasmic domains of various membrane proteins with the spectrin-based skeletal network. Band 3 and Rh-associated glycoprotein (RhAG) provide such links by interacting with ankyrin, which in turn binds to β-spectrin. Protein 4.2 binds to both band 3 and ankyrin and can regulate the avidity of the interaction between band 3 and ankyrin. Glycophorin C, band 3, XK, Rh, and Duffy all bind to protein 4.1R, the third member of the ternary junctional complex with β-spectrin and actin1–2. Red cell membrane disorders are inherited diseases due to mutations in various membrane or skeletal proteins, resulting in decreased red cell deformability, reduced life span and premature removal of the erythrocytes from the circulation. The red cell membrane disorders include hereditary spherocytosis, hereditary elliptocytosis, hereditary ovalocytosis and hereditary stomatocytosis.

Characteristic phenotypes associated with congenital dyserythropoietic anemia (type II) manifest at different stages of erythropoiesis.

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse.

Recessive hereditary methemoglobinemia: two novel mutations in the NADH-cytochrome b5 reductase gene.

We report the clinical and molecular characteristics of 6 new patients with recessive hereditary methemoglobinemia due to cytochrome b5 reductase deficiency. One patient was affected by Type-II disease with cyanosis and severe progressive neurological dysfunction, whereas the others displayed the benign Type-I phenotype. Methemoglobin levels ranged from 12.1% to 26.2% and cytochrome b5 reductase activity from 0 to 10% of normal. Eight different mutations were detected among the twelve mutated alleles identified, one splicing mutation, two stop codon, and five missense. Two mutations c. 82 C>T(Gln27STOP) and c. 136 C>T(Arg45Trp) are new. Prenatal diagnosis was performed in the family with Type-II disease.