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We describe a white Italian kindred in which hereditary elliptocytosis (HE) is associated with abnormal level of alpha I/78 peptide in spectrin digest. Clinical phenotype varied among the family members ranging from asymptomatic to mild haemolytic HE. The original mutation responsible is a G-C substitution of the spectrin alpha-gene: alpha 45 Arg-->Thr (AGG-->ACG). The corresponding spectrin is designated spectrin Anastasia. Utilizing a secondary structure predictive method we suggest that this mutation has a poor capability to induce conformational changes of the tetramerization site and thus shows a moderate elliptocytogenic potential.

This report represents an attempt to define the rate of beta-spectrin de novo mutations affecting mRNA accumulation in patients with hereditary spherocytosis (HS). 19 HS children with haematologically normal parents and varying degrees of spectrin deficiency were studied. 13 of the 19 cases who were heterozygous at the genomic level for polymorphisms in the beta-spectrin coding region were further studied. However, in an analysis of reverse-transcripted amplified cDNA from the regions of the polymorphisms, seven patients appeared to be homozygous, suggesting the occurrence of de novo mutational events affecting expression of one beta-spectrin allele. We conclude that in HS patients with isolated spectrin reduction and normal parents the apparently recessive pattern of inheritance may frequently be associated with de novo monoallelic expression of beta-spectrin.

We report three novel variants of band 3 associated with hereditary spherocytosis: band 3 Foggia (311delC; ACCCAC-->ACCAC), band 3 Napoli I (447insT; TCT-->TTCT) and band 3 Napoli II (1783N; ATC-->AAC). The first two mutations resulted in premature termination of translation, making one haploid set of band 3 mRNA unavailable. Since it affected a highly conserved position at the terminal end of transmembrane domain 11, the third mutation prevented one haploid set of band 3 from becoming incorporated or stabilized into the membrane. These three mutations resulted in a reduction of the band 3 level in the red cell membrane (by 20-25%) and were dominantly transmitted. The D38A substitution (GAC-->GCC) is a low frequency change of band 3. In one compound heterozygote D38A/Napoli II, a markedly aggravated picture required early splenectomy. In contrast, the D38A change was not associated with deterioration in another compound heterozygote, carrying in trans, the previously recorded R760W mutation (CGG-->TGG). In the aggravated case, SSCP analysis did not exhibit any additional change in the two EPB3 alleles. Nor did it show any alteration in the exons of the two ANK1 alleles, and the aggravating factor remained elusive. The D38A alteration should be regarded as an innocuous polymorphism.

We report a new mutation responsible for nonhemolytic hereditary elliptocytosis (HE). The proband displayed an impaired spectrin self- association and an increase of the alpha I 74-kD fragment (alpha I/74 abnormality). The responsible mutation occurred in exon 2 of spectrin alpha-gene: alpha 34 Arg-->Trp (CGG-->TGG), defining spectrin Genova. In Trans to allele alpha Genova, the proband disclosed allele alpha LELY, a common low-expression allele of spectrin alpha-gene. It was recognized through particular peptide maps as well as characteristic mutations in exon 40 and intron 45, respectively. The father, who carried allele alpha Genova, but not allele alpha LELY, had a milder presentation. The sensitization of allele alpha Genova by allele alpha LELY was noticeable in the proband as compared with his father. Nevertheless, it was not as sharp as that observed with many other alpha I/74 HE alleles. Therefore, each alpha I/74 HE allele has a distinct intrinsic severity.

Single point mutations of ras oncogenes are found in many tumors and contribute to the pathogenesis of the cancer. The product of the ras gene, p21 protein, was found expressed in several neuroblastoma tissues. However, the role of ras gene in this tumor has yet to be clarified. To contribute to the understanding of the ras activation, 79 fresh biopsies of neuroblastoma were studied to investigate the possibility that ras would be activated by point mutation. Analysis of H-ras and N-ras was performed by means of PCR and SSO, while K-ras mutations were detected by multiplex-ASPCR. None of the neuroblastomas examined showed H- or K-ras activation, while N-ras mutations were demonstrated in only three patients (3,7%). N-myc oncogene is amplified in a substantial number of patients with neuroblastoma. N-myc amplification was studied by Southern blot technique. N-myc amplification was demonstrated in 13.2% of patients less than 1 year of age at diagnosis and 23% of older children. Two of the patients (one stage I and one stage IVs) with N-ras mutation and without N-myc amplification had a good outcome, while the third (stage IVs) with N-myc amplification had a poor prognosis. These results suggest that ras activation is a rare event in both amplified and non-amplified neuroblastoma tumors and that N-ras activation was not involved in the clinical outcome of these patients. Moreover, our data suggest that p21 expression is induced by a post-transcriptional activation.

Alpha(I/65) Hereditary elliptocytosis (HE) is due to the duplication of TTG codon 154(1) (leucine) of alpha-spectrin and is associated with a constant haplotype. It was encountered exclusively in African and American Blacks, and in North Africans. We assumed that it diffused from the Benin-Togo area to Northern Africa. We now report two South Italian families with alpha(I/65) HE. The phenotype fully conformed to previous descriptions. The mode of transmission was dominant; however, the manifestations were more pronounced when the common, low expression level alpha(V/41) allele occurred in trans to the alpha(I/65) allele, also conforming to previous records. The mutation underlying alpha(I/65) HE turned out to be, again, the duplication of TTG codon 154 and the associated haplotype was the same as that encountered previously (+ - +; XbaI, PvuII, MspI). Thus, the alpha(I/65) allele found in Italy must have been introduced from North Africa across the Sicilian channel and would ultimately have originated from the Benin-Togo area. It would witness the same migratory stream as that followed by the Benin type haemoglobin S allele, which is also present in Southern Italy.

Recently it has been clearly established that partial deficiency of spectrin (SP) evaluated by radioimmunoassay is a common feature of many different forms of hereditary spherocytosis (HS). In this paper the determination of SP density (spectrin/band 3 ratio) in 46 HS patients and in 50 normal controls is presented. The comparison between the membrane SP density of HS subjects and controls showed a statistically significant difference (P less than 0.0005). Moreover no overlap between normal and HS subjects was observed. Membrane spectrin/band 3 ratio has been found related to some clinical features: indeed patients with severe HS showed a smaller SP density than those with milder HS. Our results show that the evaluation of membrane SP density permits a prompt diagnosis of HS and avoids extensive and unnecessary studies for other anaemias.

Background. Hereditary spherocytosis encopasses a heterogenous group of inherited disorders due to alteration of r.b.c. surface/volume ratio. Spectrin deficiency is the most common observed defect. We analyzed a case of HS associated with band 3 deficiency without spectrin reduction. Methods. In the study of a family originating from southern Italy, we show that a 20% deficiency of band 3 with normal spectrin content may be responsible for dominantly inherited hereditary spherocytosis (HS). The proband is a 12 years old girl consulting for jaundice, chronic anaemia and splenomegaly. Her mother had a similar haematologic phenotype. Results. Electrophoretic analysis of erythrocyte membrane proteins showed a deficiency in band 3 protein. Band 3 protein chymotryptic fragments, deglycosylated band 3, and its isolated cytoplasmic domain, all displayed normal electrophoretic migrations. Furthermore, the tryptic peptides profile of the cytoplasmic domain of the protein did not demonstrate any abnormality, nor did the amino acid composition of the peptides. Analysis of the membrane proteins during erythrocyte ageing, evaluated in density-fractionated red cells, showed that band 3 content was normal in the lighter fraction, whereas in the denser fraction band 3 deficiency was more pronounced than in membranes from non fractionated red blood cells. Conclusions. This case describes HS due to anion exchange transporter deficiency. Our results on fractioned red cells support the hypothesis that the defect was probably due to a band 3 protein loss during cell ageing and not to a primitive quantitative defect.

Precise data on the incidence of transforming ras oncogenes in pediatric tumors and the correlations with the histopathological properties of the tumors are very limited. Additionally the presence of ras activation in medulloblastomas has not been investigated so far. Using a combination of techniques including in vitro gene amplification by polymerase chain reaction (PCR) and detection of single base mutations by sequence-specific oligonucleotides we studied N-ras activation (mutations at codon 12, 13, and 61) in 32 medulloblastomas. DNA was isolated from 20-mu-m sections of formalin-fixed paraffin-embedded tissue. Mutations were found in 3 out of 32 examined medulloblastomas. In all cases only mutations of codon 61 were found: two of three mutations were C to A mutations at position 1 of the codon 61 (leading to a substitution of a glutamine residue for a lysine) and one was A to T mutation at position 3 in the same codon (glutamine-histidine). Our results indicate 10% incidence N-ras mutation in medulloblastoma, higher than in other CNS tumors studied so far. The main advantages of the procedure described are its greatly improved sensitivity, the increased speed with which tumor samples can be analyzed, and the possibility of using paraffin-embedded sections to analyze various rare tumors in retrospect.

We describe three italian subjects from two unrelated families affected with isolated hereditary spherocytosis (HS) without other clinical abnormalities, associated with partial spectrin and ankyrin deficiency. In both families the propositus has normal biological parents, and thus appears to be the result of a new mutation; in one of them the disease is further transmitted in an autosomal dominant fashion. Cytogenetic analysis of the latter family excluded abnormalities of the short arm of chromosome 8. We speculate that in both kindreds ankyrin deficiency is the primary defect related to ankyrin gene mutation. Several pieces of evidence suggest that ankyrin deficiency is probably the most common molecular defect in HS. It is inherited in a dominant manner and its clinical and biochemical expression is heterogenous.

Hereditary Spherocytosis (HS), or congenital hemolytic jaundice, is an important hemolytic anemia with an estimated prevalence of 1 in 5000 total live births. Approximately 75% of cases are autosomal dominant while the greater part of the remainder are inherited as recessive traits. In the last ten years, with advances in understanding of the biochemistry of the erythrocyte membrane skeleton (SDS-PAGE and IEF-SDS-PAGE after partial tryptic digestion), the molecular basis of some cases of HS have been determined. Have been found indeed deficiencies of some cytoskeleton protein as spectrin, ankyrin and band 3. The characterization of gene defects that disrupt red cell membrane protein structure and function is and will be a fascinating aim for the researchers engaged in this field.

The erythrocyte skeleton plays an essential role in determining the shape and deformability of the red cell. Disruption of the interaction between components of the red cell membrane skeleton may cause loss of structural and functional integrity of the membrane. Several observations based on studies in vitro strongly suggest that phosphorylation may modify interactions between proteins, leading to a reduced affinity. In particular, increased phosphorylation of beta-spectrin decreases membrane mechanical stability. In order to investigate the presence of membrane protein defects we investigated the erythrocyte membrane protein composition and phosphorylation in 22 children with leukemia at diagnosis and during the remission phase. Sixteen children had acute lymphoblastic leukemia (ALL), three had chronic myeloid leukemia (CML) and three had acute myeloid leukemia (AML). Ten patients (eight ALL and two CML) displayed elliptocytosis and poikilocytosis, an increase of spectrin dimers (41.8 +/- 15.6) and an enhanced phosphorylation of beta-spectrin (108 +/- 15%) at diagnosis. These alterations disappeared during the remission phase. This is the first demonstration of a reversible erythrocyte membrane alteration in leukemia. Since the beta-spectrin phosphate sites are located near the C-terminal region and close to the head of the beta-chain that is involved in dimer-dimer interaction, we supposed that the beta-chain phosphorylation has an effect upon the interactions between spectrin dimers, ie the tetramerization process. The weakening of this process should be responsible for the presence of elliptocytes and poikilocytes as reported in hereditary elliptocytosis and pyropoikilocytosis.

Objective To evaluate the frequency of de novo monoallelic expression of the ANK1 gene in hereditary spherocytosis individuals appearing as recessive. Study design We studied 40 unrelated children with spherocytosis and their normal parents. The genomic distribution of the ankyrin (AC)n dinucleotide repeats was evaluated in the patients showing combined ankyrin and spectrin deficiency. To search for the absence of mRNA derived from one of the two ANK1 genes, cDNA from the heterozygous patients was amplified using polymerase chain reaction. This was analyzed for the (AC)n dinucleotide repeats. Results Thirty-three hereditary spherocytosis subjects had variable degrees of combined ankyrin and spectrin reduction; 19 were found to be heterozygous for the AC repeat lengths and were further studied. In 12, we found a cDNA polymerase chain reaction product from one ankyrin gene alone. These findings strongly suggested the nonexpression of one of the two ANK1 genes because of the de novo mutational events. Conclusion The de novo loss of an ankyrin allele expression is a frequent cause of hereditary spherocytosis in children with normal parents. Therefore the category of genuinely recessive hereditary spherocytosis cases is further reduced compared with spherocytosis cases because of de novo mutations. The determination of the (AC)n microsatellite polymorphisms appears as a helpful and reliable tool for the discrimination between these two categories.

We describe three Italian subjects from two unrelated families affected with isolated hereditary spherocytosis (HS) without other clinical abnormalities, associated with partial spectrin and ankyrin deficiency. In both families the propositus has normal biological parents, and thus appears to be the result of a new mutation; in one of them the disease is further transmitted in an autosomal dominant fashion. Cytogenetic analysis of the latter family excluded abnormalities of the short arm of chromosome 8. We speculate that in both kindreds ankyrin deficiency is the primary defect related to ankyrin gene mutation. Several pieces of evidence suggest that ankyrin deficiency is probably the most common molecular defect in HS. It is inherited in a, dominant manner and its clinical and biochemical expression is heterogenous.

α-spectrin synthesis exceeds s by a factor four (Hanspal, 1987). This suiemeni suggests that α-spectrin deficiency would be evident only in the homozygous slate. Agre (1982, 85) described some cases of recessivcly inherited HS characterized by severe spectrin deficiency and life-threatening hemolysis. Marchcsi (1989) founded in 9 out of 20 of these kindreds an acidic shift of αII domain. More recently Forget (1990) analyzing the genomic DNA of these subjects demonstrated an Ala-Asp (GAT-GCT) substitution at position 972 of α-sp. In order to assess clinical findings of rHS and verily the linkage with α-spccirin gene we studied 11 unrelated kindreds of Italian extraction, characterized by affected propositus with hematologic ally and biochemically normal parents. Two of these had severe HS, whereas the remaining have a clinical picture of typical HS, We examined 3 polymorphic sites (Xbal, PvuII, MspI) founded in linkage disequilibrium with α-sp gene (Hoffman, 1987)(3021-EI cDNA probe was given by courtesy of B.C.Forget). In the informative families rHS is associated with the following liaplotypc X(+), P(-), M(+) in three chromosomes and with ++-, -+- and – in three others. The haplotypc ++ is the most frequent in normal population of our country. This finding suggests the heterogeneity of molecular defect at the α-gene level. Furthermore in two cases the haplotype assignement allows lo the exclusion of α-sp gene involvement. This suggests thai lite α-spectrin gene abnormalities couldn't cause all rHS.

Precise data on the incidence of transforming ras oncogenes in pediatric tumors and the correlations with the histo-pathological properties of the tumor are very limited. Additionally the presence of ras activation in medulloblastomas has not been investigated so far. Using a combination of techiques including specific in vitro gene amplification by polymerase chain reaction (PCR) (Saiki R.K., 1985) and detection of single base mutations by sequence-specific oligonucleotides (SSO) (Farr C.J., 1988) we studied N-ras activation (mutations at codon 12, 13 and 61) in 32 medulloblastomas. DNA was isolated from 5-10 μm sections of formalin-fixed paraffin-embedded tissue (according to Impraim C., 1987). Mutations were found in 3 of 32 examined medulloblastomas. In all cases only mutations of the codon 61 were found : the most frequent was a C-A at position 1 (substitution of a glutamine residue for a lysine). A mutation A-T at position 3 was present in the remaining case. The main advantage of the procedure described are its greatly improved sensitivity, the increased speed by wich tumor samples can be analysed, no longer necessary to use high-M.W DNA and the possibility to use paraffin-embedded sections to analyse various and rare tumors in retrospect.

The present paper reports the incidence from 1965 to 1979 of acute hemolytic anemia for a total of 948 cases in G-6-PD-deficient subjects due to the ingestion of fresh or dried fava beans or certain drugs and to viral infections. The highest percentage of hemolytic crises was due to fresh fava beans (94.4%). No cases of favism were observed in breast-fed babies whose mothers had eaten fava beans or from pollen inhalation. The male sex proved to be the hardest hit. Hemoglobin values were lower than or equal to 7 g/dl in about 75% of males and 50% of females. Total bilirubin values were lower than 103 mumol/l (6 mg/dl) in about 75% of males and 85% of females. Both the hemoglobin and bilirubin values were statistically significant. Mean transaminase values (SGPT) were significantly higher than those of normal controls. No correlation between favism and blood groups was found.

Glucose 6-phosphate dehydrogenase (G6PD) activity was assayed quantitatively in red cells from 100 consecutive G6PD-deficient newborn male babies born in a city hospital in Sassari, Sardinia. In four cases G6PD activity was between 30% and 45% of normal: these appeared on electrophoresis to be identical with G6PD Seattle-like. In 65 cases G6PD activity ranged from 2% to 14% of normal, while in the remaining 31 samples no activity could be detected in crude hemolysates. G6PD was partiall purified from 39 samples having activity below 14% of normal (including 20 with zero activity). G6PD activity could now be determined in all, and it was fully characterized in nine samples. These were shown to belong to two distinct classes on grounds of the Michaelis constant for glucose 6-phosphate and the elution profile from DEAE-Sephadex columns. These properties were compared with those of G6PD-deficient samples from Greece and from Israel. We conclude that there are at least three polymorphic G6PD-deficient variants in Northern Sardinia: G6PD Seattle-like, G6PD Mediterranean, and a new variant, which we designate G6PD Sassari. The GdMediterranean and GdSassari genes have been shown to breed true in family studies. We also produce evidence that the definition of G6PD Mediterranean must be carefully reassessed.

Summary Fifty mature male newborns with erythrocyte G-6-PD deficiency were used for a study con cerning the effectiveness of orotic acid in preventing severe hyperbilirubinemia. Twentyfive randomly selected neonates were given orotic acid (100 mg/kg/day) orally in two daily doses from their 1st to their 5th day of life. Twenty-five newborns were not treated According to these results orotic acid does not seem to be effective in preventing severe hyperbilirubinemia, which frequently occurs in newborn babies' with erythrocyte G-6-PD deficiency. and served as controls. Six exchange transfusions were performed in the test group and six in the controls.

Glucose-6-phosphate dehydrogenase (G6PD) electrophoretic phenotype was determined in red cells from 979 male subjects born in Naples (Southern Italy). In 0.7% of the cases no activity could be detected in haemolysates, while in 1.3% of the cases G6PD activity was approximately 20% of normal and electrophoretic mobility was altered. Moveover in two subjects a G6PD with altered mobility and normal activity was shown. G6PD was characterized in 10 subjects with variant phenotype. We conclude that the G6PD(-) phenotype in the population of Naples consists of at least six different G6PD variants associated with mild deficiency and at least one, G6PD Mediterranean, associated with severe deficiency.

We have investigated the association of neonatal jaundice (NNJ) and G6PD deficiency in consecutive births in a Northern Sardinian hospital. After excluding known causes for NNJ, and after correcting for the incidence of NNJ from unknown causes, we estimated that 20% of G6PD deficient male newborns develop NNJ resulting from their enzyme deficiency. By analyzing in detail 100 G6PD deficient babies we found no differences in birth weight or haemoglobin level between those without and those with NNJ, four of whom required exchange transfusion. We further showed by an accurate quantitative method that the residual G6PD enzyme activity was not significantly lower in G6PD deficient babies with NNJ compared to G6PD deficient babies without NNJ.