Matteo Francese

Clinical and molecular evaluation of non‐dominant hereditary spherocytosis

About 75% of hereditary spherocytosis (HS) patients have the autosomal dominant form of the disease, whereas both parents of the remaining HS patients are clinically and haematologically normal. These patients could have either the autosomal recessive form of the disease or a de novo mutation. We studied 80 randomly chosen, Italian HS children with normal parents. They had different clinical phenotypes (16 mild, 40 moderate, 16 moderately severe and eight severe). These patients were screened for the occurrence of ankyrin or β‐spectrin de novo mutations. To search for ankyrin de novo mutations affecting mRNA accumulation, we studied a (AC)n microsatellite located in the non‐coding sequence of the last exon of the ankyrin gene, and four different exonic polymorphisms in the β‐spectrin gene were utilized for the detection of de novo mutations influencing β‐spectrin mRNA stability. They were also screened for the presence of α‐spectrinLEPRA as well as for the mutation −108T→C in the ankyrin promoter, two variants previously found in some cases of genuinely recessive HS. Twenty‐five patients showed ankyrin de novo mutations and 10 HS subjects had β‐spectrin de novo mutations. Furthermore, we found five patients to be heterozygous for α‐spectrinLEPRA and one heterozygous for the mutation in the ankyrin promoter. Therefore, a molecular diagnosis was achieved in about 50% of the cases. Our data demonstrate that, among HS patients with normal parents, de novo dominant mutants are six times more common than recessive mutations. These results should be considered in view of the genetic counselling of a normal couple with a HS child.

Frequent de novo monoallelic expression of β-spectrin gene (SPTB) in children with hereditary spherocytosis and isolated spectrin deficiency

This report represents an attempt to define the rate of β‐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 β‐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 β‐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 β‐spectrin.

High frequency of de novo mutations in ankyrin gene (ANK1) in children with hereditary spherocytosis

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.

Molecular characterization of G6PD deficiency in Southern Italy : Heterogeneity, correlation genotype-phenotype and description of a new variant (G6PD Neapolis)

We report on the molecular basis of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Southern Italy (Campania region). Thirty‐one unrelated G6PD‐ deficient males were analysed at DNA level for the presence of G6PD gene mutations. Nine different G6PD variants were identified, eight of which have already been described (Mediterranean, Seattle, two different A−, Santamaria, Cassano, Union and Cosenza). G6PD Mediterranean, Santamaria, A− and Union were associated with haemolytic episodes. G6PD Seattle, which is polymorphic in several populations, Cassano and Cosenza appeared to be asymptomatic. A new variant (G6PD Neapolis) is reported here. The 467Pro→Arg substitution reponsible for G6PD Neapolis is discussed in the light of the current 3D model of human G6PD and in comparison with other natural mutations which occur in the proximity of residue 467.

Influence of methylenetetrahydrofolate reductase gene polymorphisms on the outcome of pediatric patients with non-Hodgkin lymphoma treated with high-dose methotrexate

Abstract High-dose methotrexate (MTX) is a key component of most treatment protocols for childhood and adolescent non-Hodgkin lymphoma (NHL). Recent studies have suggested that the toxicity of antifolate drugs, such as MTX, is affected by inherited single nucleotide polymorphisms (SNPs) in folate metabolizing genes. The aim of our study was to investigate the potential influence of the C677T and A1298C genetic variants of the methylenetetrahydrofolate reductase (MTHFR) gene on the clinical toxicity and efficacy of MTX in pediatric patients with NHL (n = 95) treated with therapeutic protocols Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) LNH-97 and EURO LB-02. We demonstrated that patients with the 677T genotype had an approximately six-fold greater risk of developing hematological toxicity compared with wild-type carriers, especially in the 1 g/m2 treatment group (p = 0.01). Moreover, we identified a correlation between the risk of relapse and the T genotype: T carriers had reduced disease-free survival compared with wild-type patients (67% vs. 100%). Our data suggest a pharmacogenetic influence on the adverse effects of high-dose MTX in the 1 g/m2 treatment group.

Role of polymorphic sequences 5′ to the Gγ gene and 5′ to the β gene on the homozygous β thalassemic phenotype

Sixty‐seven homozygous male and female thalassemic patients with different phenotypes, aged between 8 and 33 years, were divided into three groups, according to the severity of their β‐thalassemia (thal) mutations. We investigated whether some co‐inherited genetic factors could influence the phenotype. Patients with milder β‐thal defects, homozygotes or compound heterozygotes for the IVS‐I‐6 (T→C) or −87 (C→G) mutations had a milder disease. In addition, determination of the co‐inheritance of the −158 (C→T) Gγ polymorphism and the (AT)9T5 repeat motif in the region −540 to −525, 5′ to the β‐globin gene, showed that in some patients with severe or mild/severe β‐thal mutations, linked to haplotype III, there was higher Hb F expression. We conclude that in homozygous β‐thal patients, the severity of the mutations is the most important factor influencing the phenotype, but some polymorphisms such as the −158 (C→T) Gγ and (AT)9T5 repeat motif, increasing the Hb F expression and ameliorate the clinical course of the disease.

HEMATOLOGICAL AND MOLECULAR ANALYSIS OF β-THALASSEMIA AND Hb LEPORE IN CAMPANIA, ITALY

This epidemiological study was based on a hematological and a molecular analysis of 310 heterozygous β thalassemic and 75 carriers of Hb Lepore out of 3,000 microcythemic subjects from the Campania region of Italy. The molecular analysis of β chains and the δβ hybrid gene has shown different β chain defects, but only the Hb Lepore-Boston-Washington type in association with haplotypes I and V. The prevalence and distribution of these molecular defects in Campania show that they are linked to historical events and to the geographical characteristics of this region.

Leptin level and structure in Italian obese children

Abstract Serum leptin levels and the relationship between leptin, gender, and anthropometric measurements were assessed in a group of 40 obese and 33 lean Italian children. The coding region of OB gene was screened by polymerase chain reaction (PCR) and single strand conformational polymorphism (SSCP) for mutations in 30 unrelated obese subjects. Mean fasting leptin level was significantly higher in the obese than in the control group (18.2 ±2.1 vs 7.1 ± 1.0 ng/ml; p