Mutation identification of Fabry disease in families with other lysosomal storage disorders.

Fabry disease (FD) is an X-linked lysosomal storage disorder (LSD) caused by the deficiency of the enzyme α-galactosidase. It exhibits a wide clinical spectrum that may lead to a delayed or even missed diagnosis and the real incidence can be underestimated. We report the cases of two unrelated Italian families in whom FD was incidentally diagnosed in two females. In both families, the risk for other lysosomal disorders was known from other members affected by fucosidosis or mucopolysaccharidosis I Hurler/Scheie. Some subjects were simultaneously heterozygous for Fabry and the other lysosomal deficiency. Our study shows that the risk for more than one LSDs can occur in a family pedigree. The diagnosis of Fabry in female probands represents a diagnostic challenge, as symptoms and signs can be variably present because of the random X-chromosome inactivation.

Renal cell carcinoma of native kidney after renal transplantation: clinical relevance of early detection.

BACKGROUND: Life expectancy after transplantation has improved, and cancer may soon be the leading cause of late death after transplantation. The guidelines of the American and European societies of nephrology and urology have not yet established the optimal frequency for screening for renal cell carcinoma (RCC) of native kidneys in patients who have undergone renal transplantation. OBJECTIVE: To evaluate the prevalence, prognosis, and risk factors of RCC in a series of patients followed up for 16 years in our transplantation unit. MATERIALS AND METHODS: Our study is a follow-up observational cohort study conducted in 694 consecutive renal transplant recipients admitted to our institution from July 1991 through July 2007. At our institution, ultrasound studies of the native kidneys were performed every 6 months after renal transplantation. RESULTS: In the patient cohort studied, 10 patients developed a renal tumor (1.6% incidence). Three patients died of causes other than recurrence of RCC. Seven patients are alive with no evidence of RCC recurrence or metastatic disease after a mean (range) follow-up of 41 (12-96) months. Acquired cystic kidney disease and dialysis duration were positively associated with development of RCC. CONCLUSIONS: The incidence of RCC in the literature varies between 0.3% and 4.8%. The variability depends on the timing of follow-up, with a higher incidence in prospective studies with strict follow-up. We advise ultrasound studies performed by specialized physicians every 6 months after transplantation. More detailed guidelines designed by the major international transplantation societies are necessary.

Homozygosity for the p.K136E mutation in the SLC17A5 gene as cause of an Italian severe Salla disease.

Lysosomal free sialic acid storage diseases are recessively inherited allelic neurodegenerative disorders that include Salla disease (SD) and infantile sialic acid storage disease (ISSD) caused by mutations in the SLC17A5 gene encoding for a lysosomal membrane protein, sialin, transporting sialic acid from lysosomes. The classical form of SD, enriched in the Finnish population, is related to the p.R39C designed Salla(FIN) founder mutation. A more severe phenotype is due both to compound heterozygosity for the p.R39C mutation and to different mutations. The p.R39C has not been reported in ISSD. We identified the first case of SD caused by the homozygosity for p.K136E (c.406A>G) mutation, showing a severe clinical picture, as demonstrated by the early age at onset, the degree of motor retardation, the occurrence of peripheral nerve involvement, as well as cerebral hypomyelination. Recently, in vitro functional studies have shown that the p.K136E mutant produces a mislocalization and a reduced activity of the intracellular sialin. We discuss the in vivo phenotypic consequence of the p.K136E in relation to the results obtained by the in vitro functional characterization of the p.K136E mutant. The severity of the clinical picture, in comparison with the classical SD, may be explained by the fact that the p.K136E mutation mislocalizes the protein to a greater degree than p.R39C. On the other hand, the presence of a residual transport activity may account for the absence of hepatosplenomegaly, dysostosis multiplex, and early lethality typical of ISSD and related to the abolished transport activity found in this latter form.

The treatment of fistulae and ureteral stenosis after kidney transplantation.

INTRODUCTION: The incidence of urological complications after kidney transplantation varies from 3% to 14%, with a probable loss of the graft in 10% to 15% of cases and a mortality rate of up to 15%, despite improvements in prevention, diagnosis, and treatment as well as the use of new immunosuppressive therapies. Urinous fistulae, which are considered early complications of transplantation, are due to ischemic damage or necrosis generally occurring in the distal third of the ureter. Preservation of accessory arteries to the lower portion of the kidney is important, as they may constitute the blood supply of this segment of the collecting system or ureter. Their ligation may lead to necrosis and urinary fistulae. Ureteral stenosis, as late complication, is related to a pathology of the ureter itself, to infections, to abscesses, to fibrosis, and to ischemia. An early endoscopic approach permits resolution in 70% of cases. The aim of this retrospective study was to determine incidence and treatment of these complications. MATERIALS AND METHODS: From 1991 to 2004 we performed 453 kidney transplantations both from cadaveric and living donors. In 199 patients we performed a transvesical ureteroneocystostomy (UNCS), and in 260, an extravesical UNCS. RESULTS: The nine patients who showed fistulae (1.9%) underwent surgical treatment. In eight we used a direct ureteral reimplantation, and in one, a Boari flap technique. Nephrectomy was necessary in four patients, including two who died of septic complications. In all 26 cases of ureteral stenosis (5.6%), we used an endourological approach (anterograde or retrograde), with surgical treatment afterward in 11 patients (42%) nine direct reimplants, one anastomosis to the native ureter (transplantation from a living donor), and in one case a Boari flap technique four patients who underwent surgical treatment showed progressive damage to graft function. CONCLUSIONS: In all patients who showed fistulae we suggest surgical review: for patients with ureteral stenosis, we suggest first an endourological approach and only when it is not successful do we consider surgical treatment.

Clinical and molecular findings in patients with giant axonal neuropathy (GAN).

Giant axonal neuropathy (GAN) is a rare autosomal recessive neurodegenerative disorder of early onset, clinically characterized by a progressive involvement of both peripheral and CNS. The diagnosis is based on the presence of characteristic giant axons, filled with neurofilaments, on nerve biopsy. Recently, the defective protein, gigaxonin, has been identified and different pathogenic mutations in the gigaxonin gene have been reported as the underlying genetic defect. Gigaxonin, a member of the BTB/kelch superfamily proteins, seems to play a crucial role in the cross talk between the intermediate filaments and the membrane network. The authors report clinical and molecular findings in five Italian patients with GAN. This study shows the allelic heterogeneity of GAN and expands the spectrum of mutations in the GAN gene. The frequent occurrence of private mutations stresses the importance of a complete gene analysis.

Functional characterization of the novel mutation IVS 8 (-11delC) (-14T>A) in the intron 8 of the glucocerebrosidase gene of two Italian siblings with Gaucher disease type I.

Gaucher disease, the most common glycolipid storage disease, can be caused by a large variety of mutations. We report here the identification and characterization of a novel mutation in the human glucocerebrosidase gene, IVS 8 (-11delC) (-14T>A), in two siblings with Gaucher disease type I which occurs within the 3' end of intron 8. Both siblings were compound heterozygotes for the IVS 8 (-11delC) (-14T>A) mutation and for the c.626 G>C (R170P) substitution within exon 6. No mRNA species carrying the IVS 8 (-11delC) (-14T>A) mutation were detected by RT-PCR analysis of the RNA extracted from the patients' fibroblasts. To study the possible effects of the IVS 8 (-11delC) (-14T>A) sequence alteration on the splicing of the proximal exon 9, we have established an in vitro system generating a minigene carrying the genomic region of human glucocerebrosidase spanning from exon 8 to exon 10. Transfections into the human Hep3B cell line of the wild-type construct resulted in the expression of mRNA with the glucocerebrosidase exons correctly spliced. On the contrary, transfections of the construct carrying the IVS 8 (-11delC) (-14T>A) mutation resulted in the expression of mRNA with an 11-bp insertion located between the end of exon 8 and the beginning of exon 9. These results indicated that the 5243T>A substitution created a new 3' splice site 11 bp upstream of the wild-type one, leading to the incorporation into the mRNA of these extra 11 bases. Moreover, the new 3' splice site created by this 5243T>A transversion was preferred over the wild-type one in 100% of cases. The in vitro studies suggest that, in the patients, the 11-bp inclusion causes a shift in the reading frame with the generation of a stop codon after codon 388 which undergoes early degradation.

Molecular defects in the alpha-N-acetylglucosaminidase gene in Italian Sanfilippo type B patients.

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is a rare autosomal recessive disorder characterized by the inability to degrade heparan sulfate because of a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). We performed mutation screening in a group of 20 patients, identyifing 28 mutations, 14 of which were novel (L35F, 204delC, 221insGCGCG, G82D, W156C, 507delC, IVS3+1G-->A, E336X, V501G, R520W, S534Y, W649C, 1953insGCCA, 2185delAGA). Four of these mutations were found in homozygosity and only one was seen in two different patients, showing the remarkable molecular heterogeneity of the disease. Mutation IVS3+1G-->A produces aberrant RNA splicing: it represents a base substitution from G to A of the invariant GT dinucleotides at the splicing donor site of intron 3 resulting in the skipping of exon 3 and both exons 2 and 3. Transient transfection of COS cells, by DNA mutagenized with NAGLU mutations, produced enzymatic molecules without activity, demonstrating the deleterious nature of the defects. Metabolic labeling of transfected mutants suggested a normal synthesis of the involved polypeptide for missense alterations, whereas increased protein or mRNA instability was shown for nonsense and most of the frameshift mutations.

A novel mutation of the beta-glucocerebrosidase gene associated with neurologic manifestations in three sibs.

We report on a sibship in which three members were affected by Gaucher disease. Molecular analysis of the patients showed homozygosity for a novel mutation (C5390G) of the beta-glucocerebrosidase gene, resulting in the substitution of the arginine 353 with a glycine. Western blot analysis showed a reduced amount of beta-glucocerebrosidase-related polypeptides in fibroblasts. The phenotype resulting from this mutation is characterized by visceral and skeletal manifestations. In addition, the presence of seizures and electrophysiological abnormalities only in the 3 patients and in none of the other unaffected sibs suggests that the mutation is responsible for neurologic involvement.

Long-term complete remission after allogeneic bone marrow transplantation in multiple myeloma.

We report a case of IgG lambda type multiple myeloma treated by allogeneic bone marrow transplantation. After transplant the monoclonal protein persisted for 2 years with no other sign of disease. Thereafter the monoclonal protein was no longer detectable and the patient was considered to be in complete remission for the next 4 years.

Comparative study of 15 lysosomal enzymes in chorionic villi and cultured amniotic fluid cells. Early prenatal diagnosis in seven pregnancies at risk for lysosomal storage diseases.

A large number of chorionic villi samples obtained from women undergoing elective first trimester termination of pregnancy was analysed by enzyme assays similar to those applied to cultured amniotic cells. The levels of 15 lysosomal enzymes were compared to those observed in tissue cultures of amniotic cells obtained through amniocentesis at 16-18 weeks of pregnancy and the results were discussed in order to assess the usefulness of trophoblast biopsy for first trimester diagnosis of hereditary lysosomal diseases. The data suggest the applicability of this source of fetal cells for prenatal diagnosis of fifteen respective genetically determined enzyme deficiencies with the probable exception of alpha-L-iduronidase deficiency. Enzyme determinations were performed on chorionic villi samples of two pregnancies at risk for Tay-Sachs disease, three pregnancies for GM1 gangliosidosis type 1, one for mucopolysaccharidosis type VI and one for Wolman's disease.

[Fucosidosis: review of personal experience]. / Fucosidosi: revisione dell'esperienza personale.

Fucosidosis is a rare inherited metabolic disease characterized by severe psycho-motor degeneration. The basic defect is a deficiency of alpha-L-fucosidase which results in neuro-visceral of fucosyl compounds. We observed 7 cases of fucosidosis and an eighth case was diagnosed before birth. This review summarizes the clinical findings, radiological abnormalities and laboratory data which are indispensable for diagnosis. Clinical heterogeneity, incidence in Italian people, and difficulties in detection of carriers and in prenatal diagnosis are also discussed.