Carbon nanotubes as nanovectors for intracellular delivery of laronidase in Mucopolysaccharidosis type I.

The immobilization of proteins on carbon nanotubes (CNTs) has been widely reported mainly for the preparation of sensors while the conjugation of enzymes for therapeutic purposes has scarcely been considered. Herein we report, to the best of our knowledge, the first example of intracellular delivery of a therapeutic enzyme by means of CNTs, retaining its activity. Mucopolysaccharidosis I is a rare genetic disease characterized by the deficiency or absence of the activity of the α-l-iduronidase (IDUA) enzyme. We evaluated the capacity of the recombinant form of the human IDUA enzyme, laronidase (Aldurazyme®), conjugated with CNTs to be internalized by fibroblasts from subjects affected with Mucopolysaccharidosis type I and the capacity of the enzyme to retain its activity after internalization. The enzyme was successfully delivered into the lysosomal space and the enzymatic activity of the conjugate was preserved after internalization up to 48 hours. This paves the way towards the use of such a kind of construct for therapeutic applications.

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.

Origin and spread of a common deletion causing mucolipidosis type II: insights from patterns of haplotypic diversity.

Mucolipidosis II (ML II alpha/beta), or I-cell disease, is a rare genetic disease in which activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes, GNPTAB and GNPTG. A spectrum of mutations in GNPTAB has been recently reported to cause ML II alpha/beta. Most of these mutations were found to be private or rare. However, the mutation c.3503_3504delTC has been detected among Israeli and Palestinian Arab-Muslim, Turkish, Canadian, Italian, Portuguese, Irish traveller and US patients. We analysed 44 patients who were either homozygous or compound heterozygous for this deletion (22 Italians, 8 Arab-Muslims, 1 Turk, 3 Argentineans, 3 Brazilians, 2 Irish travellers and 5 Portuguese) and 16 carriers (15 Canadians and 1 Italian) for three intragenic polymorphisms: c.-41_-39delGGC, c.18G>A and c.1932A>G as well as two microsatellite markers flanking the GNPTAB gene (D12S1607 and D12S1727). We identified a common haplotype in all chromosomes bearing the c.3503_3504delTC mutation. In summary, we showed that patients carrying the c.3503_3504delTC deletion presented with a common haplotype, which implies a common origin of this mutation. Additionally, the level of diversity observed at the most distant locus indicates that the mutation is relatively ancient (around 2063 years old), and the geographical distribution further suggests that it probably arose in a peri-Mediterranean region.

Krabbe leukodystrophy in a selected population with high rate of late onset forms: longer survival linked to c.121G>A (p.Gly41Ser) mutation.

Krabbe leukodystrophy (KD) is a neurodegenerative lysosomal disorder caused by mutations in the galactocerebrosidase (GALC) gene. Different clinical forms are described based on the age at onset. In reported series, the early infantile form (EIKD) accounts for more than 90% of the cases. The rarer late onset forms (LOKD) become manifest later than 6 months up to the adult age. We report clinical, imaging, mutational analysis and geographic data in a large cohort of individuals with Krabbe disease examined over a 30-year period. Retrospective analyses of disease onset and long-term follow-up were conducted in 26 KD patients. Molecular analysis was performed in 12 patients and their families. Nine cases had EIKD, and 17 LOKD, accounting for two thirds of our series. No correlation was found between enzymatic activity, onset age and disease progression. Despite common geographical origin, only in a few cases could parental consanguinity be proven. The p.Gly41Ser mutation was associated with longer survival. A wide spectrum of LOKD is found despite similar genotype. Although current knowledge about onset age, residual enzyme activity and molecular analysis still fail to allow the identification of patient candidates for treatment, this information is valuable for long-term outcome prediction and could lead to reconsideration of inclusion criteria for bone marrow transplant (BMT) or other future therapeutic approaches.

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.

Type II sialidosis: review of the clinical spectrum and identification of a new splicing defect with chitotriosidase assessment in two patients.

Sialidosis is a lysosomal storage disease caused by the deficiency of alpha-N-acetyl neuraminidase-1 (NEU1). Sialidosis is classified into two main clinical variants: Type I, the milder form of the disease, and Type II, which can in turn be subdivided into three forms: congenital, infantile and juvenile. We report herein the clinical, biochemical and molecular characterisation of two patients with Type II sialidosis exhibiting the congenital (P1) and infantile forms (P2). We also review clinical data on the rare Type II forms of sialidosis in the hope of improving understanding of the disorder and facilitating its diagnosis. The genetic characterization of the two patients showed one known [c. 679G > A (p.G227R)] NEU1 missense mutation (detected in P2), and the new c.807 + 1G > A splicing defect (detected in P1), a genetic lesion that is extremely rare in this disease. Interestingly, P2 presented an extremely elevated level of chitotriosidase in plasma. This is the first pathological detection of chitotriosidase in sialidosis patients.

Enhancing cranial nerves and cauda equina: an emerging magnetic resonance imaging pattern in metachromatic leukodystrophy and krabbe disease.

We report on three cases of infantile Krabbe disease and one case of infantile metachromatic leukodystrophy showing magnetic resonance (MR) imaging findings of diffuse and coexistent cranial nerve and cauda equina nerve roots enhancement. Such findings may be simultaneous, or even precede, typical white matter abnormalities and, in the appropriate clinical context, may facilitate an earlier diagnosis. There is a rational for the use of contrast agents and craniospinal MR imaging during the first imaging of children with a history of psychomotor regression and clinical evidence of peripheral nerve involvement to exclude differential diagnoses.

Molecular and functional characterization of eight novel GAA mutations in Italian infants with Pompe disease.

We characterized 29 unrelated patients presenting with the severe form of Pompe disease (Glycogen Storage Disease Type II, acid maltase deficiency) and identified 26 pathogenic mutations divided over 28 different genotypes. Among the eight new mutations, five were exonic point mutations (c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G and c.1796C>A) leading to codon changes (p.Y191C, p.R375L, p.Q401R, p.P522A and p.S599Y); two were intronic point mutations (c.-32-3C>A and c.1636+5G>C) affecting mRNA processing; one was a single base deletion (c.742delC) generating a truncated protein (p.L248PfsX20). A comprehensive evaluation, based on different methodological approaches, confirmed the detrimental effect of the eight mutations on the protein and its function. Structural alterations potentially induced by the five missense mutations were also predicted through visual inspection of the atomic model of the GAA protein, in terms of both function and spatial orientation of specific residues as well as disturbance generated by amino acid substitutions. Although the remarkable heterogeneity of the mutational spectrum in Pompe disease was already known, our data demonstrate and confirm the power of molecular and functional analysis in predicting the natural course of Pompe disease.

Genotype-phenotype correlation in five Pelizaeus-Merzbacher disease patients with PLP1 gene duplications.

Pelizaeus-Merzbacher disease (PMD) is an X-linked myelination disorder most frequently caused by duplication of a genomic segment of variable length containing the PLP1 gene. We studied five PMD male patients affected by the classic PMD form carrying a PLP1 gene duplication. On the basis of clinical and neuroradiological features, two of the five patients appeared to be the most severely affected. In order to establish a possible genotype-phenotype correlation, the extent of the duplication was determined in each patient and in the respective mother by quantifying the copy number of genomic markers surrounding the PLP1 gene by a real-time PCR-based approach. Duplications, ranging in size from 167-195 to 580-700 kb, were in the same genomic interval of the majority of the reported duplications. The extent of the duplicated genomic segments does not correlate with the clinical severity. Interestingly enough, each duplication had one of the two breakpoints in or near to low copy repeats (LCRs), supporting recent evidence concerning a possible role of LCRs in the generation of the duplications in PMD.

An Alu insertion in compound heterozygosity with a microduplication in GNPTAB gene underlies Mucolipidosis II.

Mucolipidosis type II (ML II) is a fatal, autosomal recessive, lysosomal storage disorder characterized by severe clinical and radiologic features. ML II results from mutations in alpha and beta subunits, encoded by the GlcNAc-1-phosphotransferase gene (GNPTAB). Most of the 40 different GNPTAB mutations reported so far are insertions and deletions predicting diverse types of aberrant proteins. Alu mobile elements have however never been involved in these events up to now. The Italian ML II patient of our study showed an Alu retrotrasposition in GNPTAB exon 5. The Alu insertion mutation (NM_024312.3:c.555_556insHSU14569) generated a transcript with a skipping of the target exon 5 and a frameshift p.S122fs, causing a premature translation termination codon at position 123. This insertion mutation was found in compound heterozygosity with the frameshift p.S887KfsX33, resulting from a new mono-nucleotide duplication (c.2659dupA) that occurred in GNPTAB exon 13. A possible involvement of cis-splicing elements having an exonic location, such as exon enhancers (ESEs), is discussed as mechanism that led to the production of the aberrant mRNA splicing.

The approach by midline incision for extraperitoneal kidney transplantation.

UNLABELLED: No data are available on incisional hernia in renal transplant recipients using a midline incision. This study evaluated the incidence of abdominal wall incisional hernia, comparing two surgical approaches: midline and J-shaped incisions. METHODS: Between 1991 and 2005, 415 consecutive patients underwent renal transplantation: between 1991 and 1997, 139 patients through a lateral incision; between 1997 and 2005, 137 of 276 renal transplant patients via a midline incision, and 139 via a J-shaped incision. We evaluated the incidence of incisional herniae in these patients. Analyzed factor risks included: age, sex, body mass index, diabetes, reoperation, lymphocele, dialysis time, underlying renal disease, and immunosuppressive therapy. RESULTS: During follow-up, 15 patients of 415 transplantations were dead or lost to follow-up. Incisional herniae were identified in 12 cases of 132 (9%) between 1991 and 1997. Between 1997 and 2005 we identified 3 of 133 (2.2%) patients who underwent a midline incision and 15 of 135 (11.1%) who received a J-shaped incision (P=.005). Comparing midline and J-shaped incisions before and after 1997, the incidence reduction was significant (P=.01). Comparing the incidence among patients treated with J-shaped incision before versus after 1997, the increased incidence was insignificant (P=.6). Multivariate analysis found the most important risk factor was obesity followed by polycystic kidney disease, reoperation, wound infection, and mycophenolate mofetil therapy. CONCLUSIONS: Our data showed an advantage of a midline incision. Strategies to prevent surgical complications, such as abdominal wall relaxation and poor cosmetic results, are needed; the midline incision may be a possible alternative to address this complication.

Molecular genetics of late onset glycogen storage disease II in Italy.

Glycogen Storage Disease Type II (GSDII) is a recessively inherited disorder due to the deficiency of acid alpha-glucosidase (GAA) that results in glycogen accumulation in the lysosomes. The molecular analysis of the GAA gene was performed on 45 Italian patients with late onset GSDII. DHPLC analysis revealed 28 polymorphisms spread all over the GAA gene. Direct sequencing identified the 96% of the mutant alleles, 12 of which are novel. Missense mutations were functionally characterized by enzyme activity and protein processing in a human GAA deficient cell line while splicing mutations were studied by RT-PCR and in silico analysis. A complex allele was also identified carrying three different alterations in cis. All the patients studied carried a severe mutation in combination with a milder one, which explains the late onset of the disease. The c.-32-13T > G was the most frequent mutation, present as compound heterozygote in 85% of the patients as described in other late onset GSDII Caucasian populations. Interestingly, 10 of the 45 patients carried the c.-32-13T > G associated to the severe c.2237G > A (p.W746X) mutation. However, despite the common genotype, patients presented with a wide variability in residual enzyme activity, age of appearance of clinical signs and rate of disease progression, suggesting that other genetic/environment factors may modulate clinical presentation.

Movement and mood disorder in two brothers with Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disorder with a wide spectrum of phenotypic presentations. We report the case histories of two adult brothers with GD who developed both parkinsonism and psychiatric symptoms. Direct sequencing and real-time polymerase chain reaction were used to establish that the patients were homozygous for mutation L444P. While parkinsonism has been described previously in GD, these patients had atypical features, including a complicated mood disorder. The comorbidity of GD and a mood disorder is a new finding, as psychiatric manifestations of GD have been described rarely. The etiology of the mental illness could be related to the processes contributing to the development of parkinsonism.

GFAP mutations and polymorphisms in 13 unrelated Italian patients affected by Alexander disease.

Alexander disease (AD), a rare neurodegenerative disorder of the central nervous system, is characterized by the accumulation of cytoplasmic protein aggregates (Rosenthal fibers) composed of glial fibrillary acidic protein (GFAP) and small heat-shock proteins within astrocytes. To date, more than 40 different GFAP mutations have been reported in AD. The present study is aimed at the molecular diagnosis of Italian patients suspected to be affected by AD. By analyzing the GFAP gene of 13 unrelated patients (eight with infantile form, two with juvenile form and three with adult form), we found 11 different alleles, including four new ones. Among the novel mutations, three (p.R70Q, p.R73K, and p.R79P) were identified in exon 1 and p.L359P in exon 6. The sequence analysis also detected six different single nucleotide polymorphic variants, including two previously unreported ones, spread throughout non-coding regions (introns 2, 3, 5, 6, and 3'UTR) of the gene. All patients were heterozygous for the mutations, thus confirming their dominant effect.

Funtional characterization of four novel MAN2B1 mutations causing juvenile onset alpha-mannosidosis.

Alpha-mannosidosis is a recessively inherited disorder due to the deficiency of the lysosomal alpha-mannosidase. We report the molecular analysis performed in two patients with the late onset form of alpha-mannosidosis. Four new alleles were identified: three missense mutations involving highly conserved residues, c.597 C>A (p.H200N), c.1553 T>C (p.L518P) and c.2746 C>A (p.R916S) and a single nucleotide deletion, c.2660delC. In vitro expression studies in COS-1 cells demonstrated that pH200N, p.L518P and p.R916S proteins are expressed but retained no residual enzyme activity. These data are supported by structural 3D analysis which predicted that both p.L518P and p.R916S could affect the interaction of the small E-domain with the active site domain or the main body of the structure while the pH200N might alter substrate binding or other catalytic properties. Finally, the c.2660delC causes a frameshift introducing a premature stop codon (p.T887SfsX45), presuming to be a severe mutation.

Mutation profile of the GAA gene in 40 Italian patients with late onset glycogen storage disease type II.

Glycogen storage disease type II (GSDII) is a recessively inherited disorder due to the deficiency of acid alpha-glucosidase (GAA) that results in impaired glycogen degradation and its accumulation in the lysosomes. We report here the complete molecular analysis of the GAA gene performed on 40 Italian patients with late onset GSDII. Twelve novel alleles have been identified: missense mutations were functionally characterized by enzyme activity and protein processing in a human GAA-deficient cell line while splicing mutations were studied by RT-PCR and in silico analysis. A complex allele was also identified carrying three different alterations in cis. The c.-32-13T > G was the most frequent mutation, present as compound heterozygote in 85% of the patients (allele frequency 42.3%), as described in other late onset GSDII Caucasian populations. Interestingly, the c.-32-13T > G was associated with the c.2237G > A (p.W746X) in nine of the 40 patients. Genotype-phenotype correlations are discussed with particular emphasis on the subgroup carrying the c.-32-13T > G/c.2237G > A genotype.

Hepatic and neuromuscular forms of glycogenosis type III: nine mutations in AGL.

Glycogenosis type III (Cori disease) is an autosomal recessive disorder caused by the deficiency of the glycogen debranching enzyme, encoded by the AGL gene, and existing in six isoforms alternately spliced in a tissue-specific way. Generally, disease onset occurs early on starting from the first year of life, with hepatomegaly, hypoglycemia, hyperlipidemia, increased CK levels, and, in some cases, short stature and slight mental retardation. Frequently, hepatomegaly tends to resolve spontaneously and inexplicably during childhood, when myopathy, often associated with cardiomyopathy, arises. This disease is known to lack almost invariably clear links between the genotype and clinical phenotype. We describe nine new mutations in Italian patients: four nonsense (p.Arg285X, p.Lys422X, p.Arg910X, p.Arg977X), three frameshift (c.442delA, c.753_756delGACA, c.3963delG), and two missense (p.Ala1120Pro, p.Arg524His). Particularly, the nonsense p.Arg285X is linked to an exonic splicing enhancer and it was found to produce two species of transcripts at the same time. Moreover, we discuss a subgroup of subjects carrying c.2681+1G>A, which has proven to be the most frequent mutation among our patients. The previously described c.664+3A>G was also detected in two patients, both homozygous. The present work is yet another confirmation that the individual genetic background plays a pivotal role in influencing the phenotypes, as occurs in other metabolic diseases.

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.

Different molecular mechanisms leading to white matter hypomyelination in infantile onset lysosomal disorders.

Hypomyelinating leukoencephalopathies may be related to a primary disturbance in the formation of myelin or may be caused by neuronal, oligodendrocytic or astrocytic dysfunction, leading to a failure of myelination. Abnormal myelination related to a direct metabolic damage on oligodendrocytes has been shown to occur in some animal models of lysosomal storage diseases. To demonstrate that cerebral white matter hypomyelination may occur also in humans affected by early-onset lysosomal storage diseases, we report three cases with infantile-onset lysosomal storage disorders (type 1 GM1 gangliosidosis, globoid cell leukodystrophy or Krabbe's disease, and type A Niemann-Pick disease) showing white matter hypomyelination. Hypomyelinating leukoencephalopathy may therefore represent a feature of lysosomal storage disorders with onset in the first months of life, when the process of myelination is particularly active, indicating that neuronal storage disorders may be primarily responsible for central nervous system hypomyelination.