E Miraglia del Giudice

Ankyrin Napoli: a de novo deletional frameshift mutation in exon 16 of ankyrin gene (ANK1) associated with spherocytosis.

We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC → AAC; Asp → Asn in codon 328 of exon 10, and a one‐nucleotide deletion : CTG → CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degradation. The present allele has been designated allele Napoli.

Frequent de novo mutations of the ANK1 gene mimic a recessive mode of transmission in hereditary spherocytosis: three new ANK1 variants: ankyrins Bari, Napoli II and Anzio

A subset of spherocytosis cases associated with mutations of the ANK1 gene present an apparently recessive inheritance pattern on a clinical and haematological basis. We identified three novel out‐of‐frame deletions in the ANK1 gene: allele Bari (1361delG), Napoli II (2883delC) and Anzio (3032delCA) in three Italian patients, two of whom have been splenectomized. Analysis of the cDNA showed small or trace amounts of ankyrin mRNAs in Bari, Napoli II and Anzio. The parents were normal clinically and haematologically and did not carry the mutations exhibited by their children. We confirmed the de novo character of the HS mutations based on paternity testing. Recessive HS associated with the ANK1 gene is probably rarer than initially thought, and spherocytosis may often be due to de novo mutations.

Familial pericentric inversion of chromosome 5 in a family with benign neonatal convulsions

We describe a family in whom a pericentric inversion of chromosome 5 segregates with benign familial neonatal convulsions (BFNC). BFNC is an autosomal dominant form of epilepsy characterised by spontaneous partial or generalised clonic convulsions beginning within the first months of life. Seizures usually disappear by the age of 6 months; intercritical electroencephalogram and subsequent psychomotor development are normal. BFNC loci have been mapped to human chromosomes 20q13.3 ( BFNC1 ) and 8q24 ( BFNC2 ), based on linkage analysis.1, 2 Recently, two potassium channel genes ( KCNQ2 and KCNQ3 ), located in these two regions, were shown to be mutated in BFNC1 and BFNC2, respectively.3, 4 We report a family with BFNC and a pericentric inversion of chromosome 5 cosegregating with BFNC. Fluorescence in situ hybridisation (FISH) experiments were performed to define the breakpoints at YAC level. The linkage of BFNC to KCNQ2 and mutations in the KCNQ3 gene were excluded. This report raises the possibility of a new locus for BFNC on chromosome 5. The proband, a male, was the second child of unrelated parents, born at 40 weeks of gestation after an uneventful pregnancy. Apgar score was 10 at one and five minutes. The parents were both 32 years old at his birth. Birth weight was 3400 g (50-75th centile), length was 51 cm (50-75th centile), head circumference was 35 cm (50-75th centile), and the clinical examination was normal except for the presence of hypotelorism. …

Severe poikilocytosis associated with a de novo α28 Arg→Cys mutation in spectrin

Summary Severe poikilocytosis was observed in an Italian child. The mutation responsible was a de novoα28 Arg→Cys substitution (CGT→TGT) in spectrin, a mutation known to cause hereditary elliptocytosis or hereditary pyropoikilocytosis. In this particular case the severity of the manifestations were accounted for by the occurrence, in trans to the α28 mutation, of the αv/41 polymorphism. The latter has been shown previously to be associated with structural abnormalities at the αIV‐αV domain junction and with a low expression level. The pronounced alteration of the dimer self association process was also explained by the location of the α28 mutation. This mutation occurs in helix 3 of repeating segment α1, e.g. precisely in the head‐to‐head contact between the spectrin α and β chains. The present phenotype was compared to that yielded by another α28 mutation (Arg→His) also combined, in trans, with the αv/41 polymorphism. The pictures were very much alike, stressing the functional importance of residue α28. The de novo character of the present mutation strengthens the view that codon α28 is a ‘hot spot’ for mutations.

A novel mutation (R218Q) at the boundary between the N-terminal and the first transmembrane domain of the glycine receptor in a case of sporadic hyperekplexia

Hyperekplexia or startle disease (STHE, OMIM 149400) is a rare neurological disorder characterised by neonatal generalised muscular hypertonia followed by a period of generalised stiffness and by an exaggerated startle response to sudden external stimuli that persists throughout life.1 Hyperekplexia is usually inherited in an autosomal dominant manner although there is also evidence for a recessive form.1,2 Both forms are caused by mutations in the same gene, GLRA1 , mapping on 5q31.2 and encoding for the α1 subunit of the inhibitory glycine receptor chloride channel.1–10 Recently, it has been shown that glycine receptor beta subunit mutations can also lead to hyperekplexia.11 Glycine receptor (GlyR) belongs to the ligand gated ion channel receptor superfamily (LGICs), which includes α-aminobutyric acid, acetylcholine, and 5-hydroxytryptamine receptors and are localised in the postsynaptic membrane. All share a common pentameric structure that spans the membrane, comprising homologous subunits arranged in a ring to form a central ion conducting pore. In the case of GlyR, the 3α1–2β complex is selective for chloride ions.12 Each subunit is made up of a large glycosylated N-terminal extracellular domain, a short extracellular C-terminus, and four hydrophobic transmembrane spans (M1-M4), separated by two short hydrophilic loops M1-M2 (intracellular) and M2-M3 (extracellular) flanking the pore lining M2 domain, and by a long, extracellular, leucine rich M3-M4 loop.13,14 Considering both dominant and recessive hyperekplexia cases, 15, mostly missense, …

Reversible erythrocyte skeleton destabilization is modulated by beta-spectrin phosphorylation in childhood leukemia

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 β-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 β-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 β-spectrin phosphate sites are located near the C-terminal region and close to the head of the β-chain that is involved in dimer-dimer interaction, we supposed that the β-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.

Glucose 6-phosphate dehydrogenase deficiency and red cell membrane defects : additive or synergistic interaction in producing chronic haemolytic anaemia

Summary. We have investigated two unrelated patients with congenital haemolytic anaemia in both of whom we found a combination of hereditary spherocytosis (HS) and glucose‐6‐phosphate dehydrogenase (G6PD) deficiency. Segregation of the two defects was documented in both families, who had different molecular abnormalities for both HS and G6PD deficiency. In one family the propositus had a reduced level of spectrin and G6PD Seattle (282Asp His). In the other family the propositus had a band 3 abnormality and was heterozygous for G6PD Mediterranean (188Ser Phe). From a comparison of clinical and haematological findings in family members with either or both abnormalities we conclude that in one case the two defects exhibited a synergistic effect, resulting in a severe chronic haemolytic anaemia; whereas in the other the association was simply additive.

?I/65 Hereditary elliptocytosis in Southern Italy: Evidence for an African origin

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

Spectrin/band 3 ratio as diagnostic tool in hereditary spherocytosis.

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.

Bilirubin Levels in The Acute Hemolytic Crisis of G6PD Deficiency are Related to Gilberts Syndrome

In this study we analyzed the effect of the (TA)7 polymorphism of the UGT1A gene associated with Gilberts syndrome in G6PD-deficient subjects during an acute hemolytic crisis (fabic crisis). DNA from 44 subjects originating from the same geographic area in Sardinia was analyzed for the UGT1A promoter polymorphism. The increase of unconjugated bilirubin level during fabic crisis and its relationship with UGT1A polymorphism was evaluated. The UGT1A (TA)7 TATA box variant was found in 9/44 (21%) of the G6PD deficient subjects examined. The median value for unit of increase of bilirubin (mg/dl)/unit of decrease of hemoglobin (g/dl) was higher in variant homozygous than in heterozygous and normal subjects. These findings imply a contribution of the UGT1A polymorphism associated to Gilberts syndrome to development of the hyperbilirubinemia in G6PD deficient subjects during acute hemolytic anemia.