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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The effect of four mutations on the expression of iduronate-2-sulfatase in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome; OMIM 309900) is a rare X-linked recessive lysosomal storage disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13). Different alterations at the IDS locus, mostly missense mutations, have been demonstrated, by expression study, as deleterious, causing significant consequences on the enzyme function or stability. In the present study we report on the results of the transient expression of the novel K347T, 533delTT, N265I and the already described 473delTCC (previously named DeltaS117) mutations in the COS 7 cells proving their functional consequence on IDS activity. This type of information is potentially useful for genotype-phenotype correlation, prognosis and possible therapeutic intervention.

Efficacy and safety of Palmaz stent implantation in the treatment of renal artery stenosis in renal transplantation.

To verify the long-term efficacy and safety of Palmaz stent implantation in the treatment of transplant renal artery stenosis (TRAS), we reviewed the charts of 26 patients affected by TRAS and treated by percutaneous transluminal angioplasty (PTA) followed by permanent insertion of a Palmaz stent. The mean follow-up period was 43.31 +/- 33.6 months. The mean blood pressure fell significantly at 1 month after stenting (118 +/- 8.1 vs 101 +/- 7.8 mmHg; P < .0001); then remained stable. Renal artery blood flow, as determined by Doppler ultrasonography, was reduced from 352.5 +/- 56.5 to 157.3 +/- 53.7 cm/sec at 1 month after stenting (P < .0001). Renal function improved after stenting (serum creatinine 2.2 +/- 1.4 mg/dL preinsertion versus 1.72 +/- 1.05 at 3 years). In conclusion, in cases of severe or recurrent TRAS, stenting of the renal artery has proved to be an effective therapeutic tool. This method, which has low procedure costs and an extremely low complication rate has proved to be safe and to offer the potential of preserving luminal patency, improving the long-term efficacy of percutaneous angioplasty.

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.

Molecular analysis of 40 Italian patients with mucopolysaccharidosis type II: New mutations in the iduronate-2-sulfatase (IDS) gene.

Mucopolysaccharidosis type II (MPS2, or Hunter syndrome), rare X-linked lysosomal storage disorder, results from deleterious mutations in the iduronate-2-sulfatase (IDS) gene. We report here the mutational analysis of a total of 40 unrelated Italian MPS II patients ranging from mild to severe phenotype. We are able to assign the genotype to 29 of them (72.5%), identifying 22 different mutations, five of which are unpublished (c.533delTT, W12X, N265I, c.1131-1142del, c.1131-1305del). A total of 55.2% of the molecularly characterised patients resulted from missense mutations, 20.7% from nonsense mutations, and another 13.8% of patients from small deletions (<20pb) or splice mutations, whereas 10.3% of the cases carried major structural alterations such as large deletion and rearrangements. The results reported here support the evidence of the mutational heterogeneity of the IDS gene as well as the difficulty to correlate genotype and phenotype in the patients with MPSII. However, the molecular characterisation of the patients is advantageous, making the carrier detection feasible for the females in the family at risk and improving the reliability of prenatal diagnosis techniques. Moreover, it provides a good foundation for therapeutic strategies.

Mutational analysis of the AGL gene: five novel mutations in GSD III patients.

Total or partial lack of glycogen debranching enzyme (GDE or AGL, amylo-1,6-glucosidase, 4-alpha-glucanotransferase) is responsible for Glycogen Storage Disease type III (GSDIII), a rare autosomal recessive disorder of glycogen metabolism. The clinical and biochemical features of GSDIII subjects are quite heterogeneous, and this mirrors the genotype-phenotype heterogeneity among patients. In this paper, we report the molecular characterisation of five unrelated subjects, four Italian and one Tunisian. The following new mutations are described and confirm the genetic heterogeneity of this disease: p.R864X, p.R428K, c.3911 insA, p.G1087R and c.3512_3549dup+c.3512_3519del. The functional relevance of these mutations is discussed on the basis of the recently acquired knowledge about the boundaries and structures of the two catalytic domains.

Screening of 25 Italian patients with Niemann-Pick A reveals fourteen new mutations, one common and thirteen private, in SMPD1.

Niemann-Pick disease (NPD) results from the deficiency of lysosomal acid sphingomyelinase (SMPD1). To date, out of more than 70-disease associated alleles only a few of them have a significant frequency in various ethnic groups. In contrast, the remainder of the mutations are rare or private. In this paper we report the molecular characterization of an Italian series consisting of twenty-five NPD patients with the severe neurodegenerative A phenotype. Mutation detection identified a total of nineteen different mutations, including 14 novel mutations and five previously reported lesions. The known p.P189fs and the novel p.T542fs were the most frequent mutations accounting for 34% and 18% of the alleles, respectively. Screening the alleles for the three common polymorphisms revealed the variant c.1516G>A (exon 6) and the repeat in exon 1, but not the variant c.965C>T (exon 2). In absence of frequent mutations, the prognostic value of genotyping is limited. However, new genotype/phenotype correlations were observed for this disorder that could in the future facilitate genetic counseling and guide selection of patients for therapy.

Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations.

Mucopolysaccharidosis type I (MPS-I orMPS1) is an autosomal recessive condition characterized by a broad range of clinical symptoms. Molecular diagnosis of MPS-I is important for analyzing genotype-phenotype correlation and for selecting patients for innovative therapies. In this study we analyzed 30 Italian MPS-I patients with different phenotypes (20 severe, 6 intermediate, 4 mild) in an attempt to recognize the mutational spectrum in our population and to identify major DNA alterations specific to our country. We identified 93% of mutated alleles (56 out of 60) with the reconstruction of the complete genotype in 26 patients out of 30. Twenty-three different mutations were found, 13 of which are novel while the remaining 10 have been already described. Among the novel mutations we found 5 non conservative missense mutations (A160D, E178K, P183R, G197D, D349Y), one nonsense mutation (C53X), 6 deletions (468-470del3, 486-491del6, 755-759del5, 1251delC, 1839-1867del29, 1902-1903del2), and one splice site mutation (IVS11+5G>A). No common mutation for MPS-I is present in our country. Frequently (40% of the alleles), mutations were found in just one or two patients. However, Q70X, P533R, G51D, and W402X mutations were present in several patients (15%, 13.3%, 13.3%, and 11.6% of the alleles respectively) suggesting a Mediterranean origin of the P533R and G51D mutations. In most cases the patients' genotypes were unique combinations of mutations. The great heterogeneity found in our MPS-I population hampers mutation detection and hinders the genotype-phenotype correlation.