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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. (J Pediatr 1998;132:117-20)

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.

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.

aI/65 Hereditary elliptocytosis (HE) is due to the duplication of TTG codon 1541 (leucine) of a-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 aI/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 aV/41 allele occurred in trans to the aI/65 allele, also conforming to previous records. The mutation underlying aI/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 aI/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.

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.

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<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.

a-spectrin synthesis exceeds ß by a factor four (Hanspal, 1987). This suiemeni suggests that a-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 aII domain. More recently Forget (1990) analyzing the genomic DNA of these subjects demonstrated an Ala-Asp (GAT-GCT) substitution at position 972 of a-sp. In order to assess clinical findings of rHS and verily the linkage with a-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 a-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 a-gene level. Furthermore in two cases the haplotype assignement allows lo the exclusion of a-sp gene involvement. This suggests thai lite a-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.