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.

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.

Niemann-Pick Type C-2 Disease: Identification by Analysis of Plasma Cholestane-3ß,5α,6ß-Triol and Further Insight into the Clinical Phenotype.

INTRODUCTION: Niemann-Pick type C disease is a rare disorder caused by impaired intracellular lipid transport due to mutations in either the NPC1 or the NPC2 gene. Ninety-five % of NPC patients show mutations in the NPC1 gene. A much smaller number of patients suffer from NPC2 disease and present respiratory failure as one of the most frequent symptoms. Several plasma oxysterols are highly elevated in NPC1 and can be used as a biomarker in the diagnosis of NPC1. METHODS: Plasma cholestane-3ß,5α,6ß-triol was evaluated as biomarker for NPC2 by GC/MS and LC-MS/MS analysis. The diagnosis was confirmed by Sanger sequencing and filipin staining. RESULTS: We report three NPC2 patients with typical respiratory problems and a detailed description of the nature of the lung disease in one of them. All patients had elevated levels of plasma cholestane-3ß,5α,6ß-triol. In two of these patients, the positive oxysterol result led to a rapid diagnosis of NPC2 by genetic analysis. The phenotype of the third patient has been described previously. In this patient a cholestane-3ß,5α,6ß-triol concentration markedly above the reference range was found. CONCLUSIONS: Measurement of plasma cholestane-3ß,5α,6ß-triol enables to discriminate between controls and NPC1 and NPC2 patients, making it a valuable biomarker for the rapid diagnosis not only for NPC1 but also for NPC2 disease.The measurement of oxysterols should be well kept in mind in the differential diagnosis of lysosomal diseases, as the elevation of oxysterols in plasma may speed up the diagnosis of NPC1 and NPC2.

A nationwide survey of PMM2-CDG in Italy: high frequency of a mild neurological variant associated with the L32R mutation.

PMM2-CDG (PMM2 gene mutations) is the most common congenital disorder of N-glycosylation. We conducted a nationwide survey to characterize the frequency, clinical features, glycosylation and genetic correlates in Italian patients with PMM2-CDG. Clinical information was obtained through a questionnaire filled in by the referral physicians including demographics, neurological and systemic features, neuroimaging data and genotype. Glycosylation analyses of serum transferrin were complemented by MALDI-Mass Spectrometry (MALDI-MS). Between 1996 and 2012, data on 37 Italian patients with PMM2-CDG were collected. All the patients with a severe phenotype were unable to walk unaided, 84 % had severe intellectual disability and 81 % microcephaly. Conversely, among 17 mildly affected patients 82 % had independent ambulation, 64 % had borderline to mild intellectual disability and 35 % microcephaly. Epilepsy and stroke-like events did not occur among patients with the mild phenotype. The rate and extent of systemic involvement were more pronounced in severely affected patients. The L32R misfolding mutation of the PMM2 gene occurred in 70 % of the patients with the mild phenotype and was associated with a less severe underglycosylation of serum Tf at MALDI-MS analyses. Despite their different disease severity, all patients had progressive (olivo)ponto-cerebellar atrophy that was the hallmark clinical feature for the diagnosis. A mild neurological phenotype of PMM2-CDG marked by preserved ambulatory ability and autonomy and associated with L32R mutation is particularly frequent in Italy. PMM2-CDG should be considered in patients with even mild developmental disability and/or unexplained progressive cerebellar atrophy.

Acid sphingomyelinase: identification of nine novel mutations among Italian Niemann Pick type B patients and characterization of in vivo functional in-frame start codon.

Niemann Pick disease (NPD) is an autosomal recessive disorder due to the deficit of lysosomal acid sphingomyelinase, which results in intracellular accumulation of sphingomyelin. In the present work we studied 18 patients with NPD type B, including five individuals who presented an intermediate phenotype characterised by different levels of neurological involvement. We identified nine novel mutations in the SMPD1 gene including six single base changes c.2T>G, c.96G>A, c.308T>C, c.674T>C, c.732G>C, c.841G>A (p.M1_W32del, p.W32X, p.L103P, p.L225P, p.W244C, p.A281T) and three frameshift mutations c.100delC, c.565dupC, c.575dupC (p.G34fsX42, p.P189fsX1 and p.P192fsX14). The novel c.2T>G (p.M1_W32del) mutation inactivates the first in-frame translation start site of the SMPD1 gene and in the homozygous status causes NPD type B indicating that in'vivo translation of wild type SMPD1 initiates from the first in-frame ATG. Moreover, the new c.96G>A (p.W32X) introduces a premature stop codon before the second in-frame ATG. As a consequence of either c.2T>G (p.M1_W32del) or c.96G>A (p.W32X), impaired translation from the first in-frame ATG results in a mild NPD-B phenotype instead of the severe phenotype expected for a complete deficiency of the enzyme, suggesting that when the first ATG is not functional, the second initiation codon (ATG33) still produces a fairly functional sphingomyelinase. Analysis of the patients'clinical and molecular data demonstrated that all five patients with the intermediate phenotype carried at least one severe mutation. No association between the onset of pulmonary symptoms and genotype was observed. Finally, the presence of c.96G>A (p.W32X), the most frequent allele among Italian NPD type B population, and c.1799G>C (p.R600P) as compound heterozygotes in association with severe mutations suggested a beneficial effect for both mutations.

Intractable fever and cortical neuronal glycogen storage in glycogenosis type 2.

Glycogenosis type 2 is an autosomal recessive glycogen storage disorder caused by deficiency of lysosomal acid alpha-glucosidase. Different phenotypes are recognized. The authors describe two children affected by the late infantile form; both presented terminal hyperthermia not caused by infections. Autopsy performed in one case showed diffuse glycogen storage in the CNS neurons. In light of current interest in enzyme replacement therapy, this finding casts some doubt on how effective enzyme replacement therapy will be unless it can be targeted directly into the CNS.

Infantile sialic acid storage disease: biochemical studies.

Infantile free sialic acid storage disease (ISSD), is an inherited metabolic disorder characterized by hyperexcretion of free sialic acid in the urine and by its storage in the lysosomes of different tissues. In order to obtain more reliable data on the amount of total and free sialic acid, we analyzed the urine, brain, cerebellum, liver, spleen, and kidneys from a 3-month-old baby who died with a diagnosis of ISSD. The lysosomal nature of the disease was confirmed by an electron microscopic study of cells in culture. No significant abnormalities were found involving cholesterol, total phospholipids, glycolipids, and gangliosides in the tissues examined. However, differences in the tissue distribution of individual glycolipids and gangliosides were observed. The amount of free and total sialic acid was markedly increased, due to the storage of free sialic acid accompanied by its hyperexcretion in the urine. These results demonstrate and confirm that only acid monosaccharide transport from the lysosome compartment is involved in the pathogenesis of ISSD.

Bone complications in children with Gaucher disease.

For paediatric patients with Gaucher disease, enzyme replacement therapy (ERT) has the potential to prevent the development of serious, irreversible skeletal complications. Analysis of skeletal data for paediatric patients receiving ERT must take into account the pubertal growth spurt and developmental changes in bone marrow composition. In a study conducted at the Burlo Garofolo Institute in Trieste, Italy, 10 paediatric patients have received ERT, and data are available for 3-9 years of follow-up. ERT was associated with a significant increase in the mean lumbar bone mineral density (BMD) Z score after 2 years of treatment (p=0.003). Skeletal growth rates increased among patients exhibiting growth delays. At the Gaucher Disease Treatment Center in Cincinnati, OH, USA, a total of 11 paediatric patients have been followed for 2 years or more of ERT. Of these 11 patients, 6 have demonstrated significant increases in lumbar BMD after 2 years of ERT; these patients tended to have lower BMD Z scores at the start of ERT. At the Children's Hospital of the Johannes-Gutenberg University in Mainz, Germany, 7 children with type 1 Gaucher disease presented with reduced BMD in the distal ulna, and after 18-24 months of ERT, these patients demonstrated increases in BMD at this site. The patients exhibiting growth retardation experienced growth acceleration during treatment. These studies suggest that ERT improves BMD and growth rates in paediatric patients with Gaucher disease. ERT in paediatric patients may have the potential to prevent serious skeletal complications such as fractures and vertebral compression later in life.

Response of Gaucher bone disease to enzyme replacement therapy.

In Gaucher disease, enzyme replacement therapy usually reduces liver and spleen volumes and improves haematological abnormalities within 1 year. In contrast, skeletal manifestations of Gaucher disease are thought to respond more slowly. For example, decreased bone marrow glycolipid infiltration and increased bone mineral density have been reported to take up to 3-4 years of treatment. In this report, we present recent studies using T1- and T2-weighted MRI and quantitative chemical shift imaging that demonstrate decreases in abnormal glucocerebroside infiltration and increases in normal fat content of bone marrow within the first year of treatment. There was no obvious relationship between age, gender, splenectomy status or genotype and the response of bone marrow to therapy. Although the dose of enzyme replacement therapy may be related to bone marrow response, no significant relationship was demonstrated in this report. Long-term enzyme replacement therapy induces continued degradation of Gaucher cell deposits, reconversion of fat marrow and increased bone mineral density. This treatment is also associated with improved or non-progressive bone symptoms and functional status in most adult patients, and it prevents the new occurrence of bone pain and bone crisis in nearly all patients. The development of more sensitive, quantitative imaging methods will help to evaluate disease severity better and to assess the response to therapy.

Treatment of patients with Niemann-Pick type is using repeated amniotic epithelial cells implantation: correction of aggregation and coagulation abnormalities.

Variations of platelet aggregation and plasma levels of clotting factors V, IX, XI, and XII were studied in 5 patients with Niemann-Pick disease type Is in the course of a 3-year study of treatment with periodic subcutaneous infusions of amniotic epithelial cells. Before commencement of treatment, the concentrations of these factors were found to be abnormal in four of five patients. It was possible to complete the study protocol in only two patients. Platelet aggregation and plasma levels of V, IX, XI, and XII clotting factors had been determined before each epithelial amniotic cells implantation and after 24, 48, and 72 hours. In both patients the aggregation test and the plasma levels of coagulation factors V, IX, XI, and XII were below the normal values of reference. Results showed that the epithelial amniotic cells treatment normalized platelet aggregation after each implantation in the two studied patients, both in terms of intensity of response (increase in light transmission after addition of adenosine diphosphate up to 350%) and in terms of obtaining an irreversible aggregation with 3 and 8 microM of adenosine diphosphate. The data related to clotting factors showed an increase of these concentrations up to 60% and some of these concentrations normalized completely.

[Hypertrophic stenosis of the pylorus. Correlations with allergy to milk proteins and atopy]. / Stenosi ipertrofica del piloro. Correlazioni con l'allergia alle proteine del latte e con l'atopia.

We performed a study concerning the relationship between hypertrophic pyloric stenosis (HPS), atopy and cow's milk protein allergy (CMPA). Familial history of atopy was documented in 17 (44.7%) of 38 infants having undergone Ramstedt pyloromyotomy for HPS, in 12 (23%) of 52 infants having undergone surgery for inguinal hernia and in 53 (26.9%) of 290 normal controls (significant differences between HPS and other groups). Moreover 9 (23.6%) of 38 infants with HPS presented personal history of eczema at follow-up compared with 3 (5.7%) infants having undergone surgery for inguinal hernia (p less than 0.01). A significant difference between HPS and other groups was also found when looking for familial history of CMPA: 26.3% vs % and 2.5% respectively (p less than 0.001). Eventually we discovered a higher than expected incidence of CMPA both in a retrospectively evaluated group of HPS and in 24 infants undergone Ramstedt pyloromyotomy and followed according to a standardized protocol for a mean period of 4 months (16.6%). The etiology of HPS remains obscure. Our data however suggest some relationship between HPS, atopy and particularly CMPA.

Premature birth with complicated perinatal course delaying diagnosis of prader-willi syndrome.

Prader-Willi syndrome in the newborn is essentially characterized by marked hypotonia, feeding difficulties, hypogonadism, and possible characteristic facial features. However, diagnosis at this age may be particularly difficult, and dysmorphic features may be subtle or absent. Prematurity can furthermore delay clinical features recognition and typical complications due to preterm birth may contribute to divert the diagnosis. We describe a preterm baby with a complicated perinatal course later diagnosed as PWS.

Substrate reduction therapy in the infantile form of Tay-Sachs disease.

Substrate reduction therapy (SRT) with miglustat has been proposed for treatment of some lysosomal storage disorders. Based on the positive experience in Gaucher disease and experimental data in Tay-Sachs (TSD) and Sandhoff animal models, the authors investigated the clinical efficacy of SRT in two patients with infantile TSD. SRT could not arrest the patients' neurologic deterioration. However, a significant drug concentration in CSF as well as macrocephaly prevention were observed.

Gaucher disease and bone: laboratory and skeletal mineral density variations during a long period of enzyme replacement therapy.

The usefulness of bone turnover markers in Gaucher disease is still unclear and their utility in monitoring the effects of enzyme replacement therapy (ERT) on bone metabolism has not yet been investigated exhaustively. Skeletal involvement seems to improve slowly during ERT, but only a few studies evaluating bone mineral density (BMD) changes during a long follow-up period have been reported. The aim of this study was to assess the efficacy of ERT on bone involvement in a group of 12 type I Gaucher disease (GD I) patients by monitoring biochemical indices of bone resorption/formation and BMD measured by dual energy x-ray absorptiometry (DEXA). Serum (calcium, phosphorus, bone alkaline phosphatase isoenzyme, carboxyterminal propeptide of type I procollagen (PICP), carboxyterminal telopeptide of type I collagen (ICTP), osteocalcin, intact parathyroid hormone) and urinary (calcium, phosphorus, hydroxyproline and free deoxypyridinoline) markers of bone metabolism and lumbar BMD were measured at baseline, after 6 and 12 months, and then every year for a mean ERT follow-up period of 4.5 years (range 4.4-6 years). Twelve healthy adult subjects matched for age and sex were tested as negative controls. A significant decrease of PICP was detected in the patient group at baseline (mean value 100.52 ng/ml vs 142.45 ng/ml, p = 0.017), while ICTP was remarkably higher: mean value 3.93 ng/ml vs 2.72 ng/ml, p = 0.004 (two-sided Student's t-test). No changes in bone formation indices were observed during the follow-up period, while urinary calcium excretion increased significantly from 0.065 to 0.191 mg/mg creatinine (p = 0.0014) (repeated measures ANOVA). A significant BMD improvement was also detected after an average ERT period of 4.5 years: Z-score increased from -0.81 to -0.56 (p = 0.005) (two-sided Student's t-test). These data evidenced the ineffectiveness of the biochemical markers used in monitoring ERT efficacy in GD I skeletal involvement, whereas DEXA was demonstrated to be a reliable method with which to follow up BMD improvement.

[Efficacy of magnesium in a case of neonatal pulmonary hypertension refractory to the usual therapies]. / Efficacia del magnesio in un caso di ipertensione polmonare neonatale resistente alle comuni terapie.

We describe a premature baby with X pentasomy, having a severe pulmonary hypertension secondary to perinatal asphyxia and hyaline membrane disease which appeared to be refractory to conventional treatments (hyperventilation, tolazoline, prostacyclin). Oxygenation and pulmonary hypertension rapidly improved after starting magnesium sulphate infusion (loading dose: 20 mg/kg e.v. of elementary Mg, followed by continuous infusion of 2-4 mg/kg/hr during 6 days). The therapy was associated with hypermagnesemia (5-7 mg/100 ml) and transitory side effects (hypocalcemia, muscular and bladder paresis, bradycardia without hemodynamic decompensation). We suggest that magnesium therapy might be considered in newborns with severe and persistent pulmonary hypertension.

The effect of bacterial infection in the worsening of atopic dermatitis: correlations with humoral immunologic patterns.

The contribution of secondary infection to severity and tendency to relapse in atopic dermatitis during childhood has been assessed. A total of 57 children aged between 4 months and 14 years were followed for an average of 4.73 months. A secondary infection was diagnosed in 22 (31.4%) of 70 relapses, since the lesions only subsided with antibiotics active on the bacteria isolated from the skin, usually a coagulase-positive Staphylococcus aureus. The eczema was more severe at presentation and hypogammaglobulinemia G more often found in those children who were more susceptible to secondary infections. The hypogammaglobulinemia G was present in 13 out of the 57 patients, but it normalized with age and was not correlated with IgE levels. In the children in whom the relapse or the worsening of the eczema could be attributed to secondary infection because of the positive response to the antibiotic treatment, the lesions had the appearance of pustules or showed more exudation, although in some cases only the worsening of the erythema and itching was observed. A secondary bacterial infection should be considered a likely cause of relapse or worsening of atopic dermatitis. Furthermore it may be that, at least in first year of life, hypogammaglobulinemia G is part of an immunologic impairment of atopic dermatitis which favors the susceptibility to secondary infections.

Idiopathic arterial calcification of infancy: effectiveness of prostaglandin infusion for treatment of secondary hypertension refractory to conventional therapy: case report.

A premature baby had severe hypertension associated with idiopathic arterial calcification of infancy. Despite the fact that there was laboratory evidence of renin-mediated hypertension, the disease was refractory to specific renin antagonist and failed to respond to conventional medical treatment. Prostaglandin E1 (PGE1) infusion (dosage range 0.017-0.068 microgram/kg/min) promptly controlled hypertension on two occasions. The drug was given for a total of 65 days and then stopped after the appearance of severe thrombocytopenia; other side effects included sporadic hyperthermia and irritability. Blood pressure was then stabilized satisfactory by a multiple-antihypertensive regimen. In the light of these findings, we believe that PGE1 infusion is a possible therapeutic alternative for babies with idiopathic arterial calcification complicated by severe hypertension refractory to conventional treatment.

Intravenous pamidronate treatment in osteogenesis imperfecta.

Three children with osteogenesis imperfecta, severe osteopenia, and repeated fractures were treated with cyclic infusions of aminohydroxypropylidene bisphosphonate (pamidronate) for a period ranging from 22 to 29 months. A clear clinical response was shown, with a striking reduction of new fracture episodes and a marked improvement in the quality of the patients' lives. Bone mineral density increased significantly in two patients, and linear growth continued along the percentile at the start of treatment. There were no adverse effects of note during treatment, and further studies are warranted.

Bone marker alterations in patients with type 1 Gaucher disease.

Bone involvement is one of the most disabling aspects of type I Gaucher disease and its pathophysiology is still not well understood. As an invasive procedure, bone biopsies are not appropriate in a large population study. The development of sensitive bone resorption and formation tests have allowed the authors to study bone metabolism in a noninvasive manner in a group of type 1 Gaucher patients. Ten type I Gaucher adult patients with mild-to-severe bone disease were evaluated. Bone mineral density and markers of bone formation (total alkaline phosphatase and isoenzymes, carboxyterminal propeptide of type I procollagen, osteocalcin) and resorption (carboxyterminal telopeptide of type I collagen, urinary hydroxyproline, free-deoxypyridinoline and calcium) were measured in patients and in a control group, matched for sex and age. In Gaucher patients, carboxyterminal propeptide of type I procollagen (PICP), a bone formation index, was significantly lower compared with normal subjects (mean 101.17 ng/ml vs 140.75 ng/ml, P = 0.038), and analysis of bone resorption indexes showed a significant increase (mean 4.24 ng/ml vs 2.87 ng/ml, P = 0.012) of serum carboxyterminal telopeptide of type I collagen (ICTP). No significant differences were observed in osteocalcin, alkaline phosphatase, and urinary hydroxyproline. Bone mineral density revealed osteopenia in six patients, with a mean Z-score of ?1.04. It was not possible to show a relationship between sex, splenectomy status, age, weight, spleen, and liver volume and bone density, expressed as a Z-score nor a correlation between Z score and severity of skeletal disease. Results have shown a predominance of the resorption phase in the bone metabolism of Gaucher patients. These markers could be useful in monitoring the effect of enzyme replacement therapy on Gaucher disease skeletal involvement.

Effectiveness of adjunctive treatment with steroids in reducing short-term mortality in a high-risk population of children with bacterial meningitis.

Bacterial meningitis is still an important cause of death and/or persistent nervous system damage in children living in developing countries. The aim of the study was to evaluate the effectiveness of steroids in reducing mortality and neurologic sequelae in children affected by bacterial meningitis within the context of a developing country (Mozambique), where the case-fatality rate of this disease is over 30 per cent. Seventy children with bacterial meningitis were randomized to receive either conventional antibiotic therapy or antibiotic therapy plus dexamethasone. On hospital admission there were no statistically significant differences between the two groups with regard to clinical and laboratoristic features. When dexamethasone was used early mortality, within 24 h, was significantly reduced (1/34 v. 8/36, P < 0.05). Total mortality among steroid treated patients, including those who were comatose on admission, was also reduced even if the difference did not reach statistical significance. A favourable trend in terms of fewer serious neurologic abnormalities was also observed among survivors in the steroid treated patients (5/26 v. 7/24). Fever and CSF abnormalities also disappeared more rapidly in patients receiving dexamethasone (P < 0.05). This study showed that the beneficial effect of adjunctive steroid therapy in children with bacterial meningitis can be even more important in areas where the case-fatality rate of this disease is still very high.