Red cell pyruvate kinase deficiency in Southern Sardinia.

Pyruvate kinase (PK) deficiency is the most frequent red cell enzymatic defect responsible for hereditary non-spherocytic hemolytic anemia. The clinical picture is quite variable and the reasons of this variability have been only partially clarified. We report the clinical description and the extended molecular analysis in 3 PK deficient patients with clinical phenotype of variable severity. We studied the clinical and hematological aspects of 3 patients and analyzed the following genes: pyruvate kinase-R, glucose-6-phosphate-dehydrogenase, α-globin, uridindiphosphoglucuronil transferase and HFE. One patient (A) with a severe clinical picture resulted homozygote for exon 8 nt994A substitution, the other 2 (brothers) were compound heterozygotes for exon 8 nt994A and exon 11 nt1456T mutation. One of the two brothers with a more severe phenotype coinherited also had G6PD deficiency, while both had microcytosis due to the homozygosity for the non-deletional form of α-thalassemia ATG→ACG substitution at the initiation codon of the alpha2 globin gene. Our results suggest that extended molecular analysis is useful for studying how several interacting gene mutations contribute to the clinical variability of pyruvate kinase deficiency.

Thalassaemia and glucose-6-phosphate dehydrogenase screening in 13- to 14-year-old students of the Sardinian population: preliminary findings.

OBJECTIVES: In this paper we describe the outline and results of a 7-year screening programme for thalassaemias and glucose-6-phosphate dehydrogenase (G6PD) deficiency in 13- to 14-year-old students from the Sardinian population. METHOD: This programme had several steps: formal education on thalassaemia, request of informed consent by parents, blood testing and genetic counselling. RESULTS: Out of 63,285 subjects tested, 6,521 (10.3%) were heterozygotes for beta-thalassaemia, 16,175 (25.6%) for alpha-thalassaemia and 101 were carriers of a haemoglobin variant. One thousand four hundred and twenty (16.4%) males were hemizygotes for G6PD deficiency and 1,893 (20.6%) females were heterozygotes. CONCLUSION: The uptake of the programme was remarkably high and homogeneous across the island, indicating and confirming a great interest of the Sardinian population in any initiative directed at the prevention of homozygous beta-thalassaemia.

Identification of a novel T-insertion polymorphism at the DMD locus.

We have identified a novel T-insertion polymorphism located in the second intron of the dystrophin gene. This polymorphism should prove useful in linkage studies in Duchenne and Becker muscular dystrophy families in addition to the previously described markers.

Germinal mosaicism in a Duchenne muscular dystrophy family: implications for genetic counselling.

In this study we describe a three-generation family in which two siblings were affected by Duchenne muscular dystrophy (DMD). Immunohistochemical analysis of muscle dystrophin and haplotype analysis of the DMD locus revealed that the X chromosome carrying the DMD gene was transmitted from the healthy maternal grandfather to his three daughters, including the proband's mother. These findings indicate that the grandfather was a germinal mosaic for the DMD gene. The definition of the carrier status in two possible carriers led us to give accurate genetic counselling and to prevent the birth of an affected boy. The results of this study demonstrate the usefulness of haplotype analysis and immunohistochemical muscle dystrophin studies to detect hidden germinal mosaicism and to improve genetic counselling.

Diagnosis of DMD carrier status in a family with no known affected males.

A 30-year-old woman and her two-year-old daughter were found by chance to have moderately raised serum creatine kinase (CK) levels. Since the mother was pregnant, the authors investigated the possibility that the two females were carriers of the common Duchenne muscular dystrophy (DMD) gene. No immunohistochemical abnormality was detected in the mother, but in the daughter a clear mosaic pattern of dystrophin positive and negative fibres was found, indicating carrier status for DMD. These data indicate that a diagnosis of DMD carrier status can be made even in families without a positive history for this disorder; therefore, immunocytochemical studies, using antidystrophin antibodies, should be performed on all females with raised CK levels, including the youngest.

Dystrophin analysis using a panel of anti-dystrophin antibodies in Duchenne and Becker muscular dystrophy.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in 19 patients with Xp21 disorders and in 25 individuals with non-Xp21 muscular dystrophy. Antibodies raised to seven different regions spanning most of the protein were used for immunocytochemistry. In all patients specific dystrophin staining anomalies were detected and correlated with clinical severity and also gene deletion. In patients with Becker muscular dystrophy (BMD) the anomalies detected ranged from inter- and intra-fibre variation in labelling intensity with the same antibody or several antibodies to general reduction in staining and discontinuous staining. In vitro evidence of abnormal dystrophin breakdown was observed reanalysing the muscle of patients, with BMD and not that of non-Xp21 dystrophies, after it has been stored for several months. A number of patients with DMD showed some staining but this did not represent a diagnostic problem. Based on the data presented, it was concluded that immunocytochemistry is a powerful technique in the prognostic diagnosis of Xp21 muscular dystrophies.

Variable dystrophin expression in different muscles of a Duchenne muscular dystrophy carrier.

The majority of Duchenne muscular dystrophy (DMD) female carriers show dystrophin immunostaining abnormalities, although a significant proportion of clinically non-manifesting carriers are normal following this analysis. We had the opportunity to study dystrophin immunostaining in two different muscles, the vastus lateralis and the rectus abdominis of a possible DMD carrier. While the vastus showed normal dystrophin immunostaining, pathological staining was detected in her rectus abdominis. These findings seem to indicate that dystrophin expression can vary in different muscle groups of a DMD carrier. The implications of these findings in DMD carrier detection and possible dystrophin function are discussed.

Mesangiolysis and endothelial lesions due to peroxidative damage in rabbits.

There is much evidence that oxygen free radicals (OFR) may be the final mediators of biochemical and molecular damage in many kidney diseases of different etiology (toxic, ischemic and immunologically mediated), involving mainly endothelium, basement membrane and tubular cells, but direct demonstration of a role in inducing mesangiolysis is lacking. An experimental model of renal damage caused by OFR was carried out in 6 rabbits using a mixture of xanthine-oxidase and xanthine, which produces a large amount of the radical superoxide anion. Both enzyme (0.0150 and 0.150 U/ml) and substrate (0.2 and 2 mM) were simultaneously infused in one kidney, while the controlateral kidneys perfused with buffer only were used as controls. Treated kidneys were compared to controls by light and electron microscopy. A further experiment was carried out in 4 other rabbits to evaluate the protection afforded by superoxide dismutase, the specific enzyme-scavenging superoxide anion. Microscopic studies showed dose-related ingravescent damage in the treated kidneys: capillary enlargement, subendothelial swelling, detachment of the endothelium from the basement membrane, mesangiolysis and microaneurysms. Control kidneys appeared to be normal. No significant differences were observed in the kidneys treated with addition of superoxide dismutase. These results are the first direct demonstration of a role of superoxide anion in the induction of mesangiolysis in rabbits. The lack of a protective effect by superoxide dismutase could mean that the superoxide anion triggers a chain of other OFR, further responsible for damage.