Elisabetta Pasquini

North Star Ambulatory Assessment, 6-minute walk test and timed items in ambulant boys with Duchenne muscular dystrophy

The North Star Ambulatory Assessment is a functional scale specifically designed for ambulant boys affected by Duchenne muscular dystrophy (DMD). Recently the 6-minute walk test has also been used as an outcome measure in trials in DMD. The aim of our study was to assess a large cohort of ambulant boys affected by DMD using both North Star Assessment and 6-minute walk test. More specifically, we wished to establish the spectrum of findings for each measure and their correlation. This is a prospective multicentric study involving 10 centers. The cohort included 112 ambulant DMD boys of age ranging between 4.10 and 17 years (mean 8.18±2.3 DS). Ninety-one of the 112 were on steroids: 37/91 on intermittent and 54/91 on daily regimen. The scores on the North Star assessment ranged from 6/34 to 34/34. The distance on the 6-minute walk test ranged from 127 to 560.6 m. The time to walk 10 m was between 3 and 15 s. The time to rise from the floor ranged from 1 to 27.5 s. Some patients were unable to rise from the floor. As expected the results changed with age and were overall better in children treated with daily steroids. The North Star assessment had a moderate to good correlation with 6-minute walk test and with timed rising from floor but less with 10 m timed walk/run test. The 6-minute walk test in contrast had better correlation with 10 m timed walk/run test than with timed rising from floor. These findings suggest that a combination of these outcome measures can be effectively used in ambulant DMD boys and will provide information on different aspects of motor function, that may not be captured using a single measure.

GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.

The inclusion of succinylacetone as marker for tyrosinemia type I in expanded newborn screening programs

In expanded newborn screening programs by liquid chromatography/tandem mass spectrometry false negatives for tyrosinemia type I are a significant problem. We describe a method for inclusion of succinylacetone in order to avoid false negatives. We studied spots from 13,000 neonates born in Tuscany (January-May 2007) and ten spots from six patients with tyrosinemia type I. The traditional screening method was modified by adding dioxooctanoid acid (or 13C2-succinylacetone) as an internal standard to the methanolic solution of deuterated acylcarnitines and amino acids. A hydrazine solution was added to the mixture. The times of extraction, butylation and drying were only slightly prolonged. Specific multiple reaction monitoring for derivatized and labelled succinylacetone and dioxooctanoic acid was carried out. The assays were linear up to 100 micromol/L for succinylacetone. Intra- and inter-day imprecision data were in the range of 1.34% to 7.09% and 3.50% to 4.49%. Limits of detection and of quantification were 0.2 micromol/L and 0.4 micromol/L, respectively. Recovery ranged from 97.02% to 100.29%. Succinylacetone levels in samples from unaffected neonates were very close to the detection limit. Of the 46 recalls, eight (17.4%) were for abnormal tyrosine levels and all these cases had succinylacetone levels within the normal range (<2.4 micromol/L). In ten spots from six affected patients succinylacetone values ranged from 3.3 to 35.0 micromol/L. Including succinylacetone in newborn screening programs for amino acids and acylcarnitines avoids false-negative results for tyrosinemia type I. Newborn screening laboratories should consider implementing these modifications.

Newborn screening for homocystinurias and methylation disorders: systematic review and proposed guidelines

Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency.

Rapid diagnostic testing procedures for lysosomal storage disorders : α-glucosidase and β-galactosidase assays on dried blood spots

BACKGROUND Lysosomal storage disorders (LSDs) are pathologies caused by the deficit of lysosomal enzymes; late diagnosis may render therapeutic programs less effective. As early, pre-symptomatic detection could change the natural history of the disease, we are setting up rapid microassays using dried blood spots (DBS) on filter paper. Here we report alpha-glucosidase and beta-galactosidase assays. METHODS Enzymatic activities were evaluated on DBS from five different groups of subjects including healthy controls and patients affected with an LSD. A 260-day monitoring of DBS preservation at five different temperatures and a comparison of the enzymatic activities measured in DBS obtained from a single (sDBS) or a double (dDBS) blood drop were performed as well. RESULTS Both assays could clearly distinguish the affected patients from the other subjects analyzed. Storage of DBS at 4 degrees C and below allowed a longer preservation of the enzymatic activities. No significant differences were detected between sDBS and dDBS. CONCLUSIONS DBS can be used for non-invasive, easy, inexpensive lysosomal enzyme assays. Reliability of assays on DBS needs to be checked using a control enzyme such as beta-galactosidase. DBS can be still reliably analyzed even if generated incidentally by two overlapped drops.

3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

BackgroundIsolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and β subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults.MethodsWe report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby.ResultsFifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date.ConclusionsOur data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.

Genetic polymorphisms of antioxidant enzymes as risk factors for oxidative stress-associated complications in preterm infants

Abstract We aimed to identify specific polymorphisms of genes encoding for superoxide dismutase (SOD) 1 (cytoplasmic Cu/ZnSOD), SOD2 (mitochondrial MnSOD), SOD3 (extracellular Cu/ZnSOD) and CAT in a cohort of preterm infants and correlate their presence to the development of respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), intraventricular haemorrhage (IVH) and retinopathy of prematurity (ROP). We carried out a retrospective study to evaluate the allele frequency and the genotype distribution of polymorphisms of SODs and CAT in a population of preterm neonates (n = 152) with a gestational age ≤ 28 weeks according to the presence or absence of RDS, BPD, IVH and ROP. Moreover, we evaluated through the haplotype reconstruction analysis whether combinations of the selected polymorphisms are related to the occurrence of RDS, BPD, IVH and ROP. We found that rs8192287 SOD3 polymorphism is an independent protective factor for all grade IVH, while rs4880 and rs5746136 SOD2 polymorphisms are associated with a lower gestational age (rs4880, rs5746136) and birth weight (rs4880). Haplotypes reconstruction showed that SOD1 (GG) decreased the risk of RDS, IVH and ROP; SOD2 (GT) increased the risk of BPD and decreased the risk of RDS, IVH and ROP; SOD3 (TGC) decreased the risk of BPD and IVH; and 4) CAT (CTC) decreased the risk of RDS. The rs8192287 SOD3 polymorphism is per se an independent predictor of a decreased risk of developing IVH. Different SOD2 polymorphisms are associated per se with a lower gestational age and/or birth weight, and haplotypes of SOD1, SOD3 and CAT genes may be independent protecting or risk markers for prematurity complications.

The de novo Q167K mutation in the POU1F1 gene leads to combined pituitary hormone deficiency in an Italian patient.

The POU1F1 gene encodes a transcription factor that is important for the development and differentiation of the cells producing GH, prolactin, and TSH in the anterior pituitary gland. Patients with POU1F1 mutations show a combined pituitary hormone deficiency with low or absent levels of GH, prolactin, and TSH. Fourteen mutations have been reported in the POU1F1 gene up to now. These genetic lesions can be inherited either in an autosomal dominant or an autosomal recessive mode. We report on the first Italian patient, a girl, affected by combined pituitary hormone deficiency. The patient was found to be positive for congenital hypothyroidism (with low TSH levels) at neonatal screening. Substitutive therapy was started, but subsequent growth was very poor, although psychomotor development was substantially normal. Hospitalized at 10 mo she showed hypotonic crises, growth retardation, delayed bone age, and facial dysmorphism. In addition to congenital hypothyroidism, GH and prolactin deficiencies were found. Mutation DNA analysis of the patients POU1F1 gene identified the novel Q167K amino acid change at the heterozygous level. The highly conserved Q167 residue is located in the POU-specific domain. No mutation was detected in the other allele. DNA analysis in the probands parents did not identify this amino acid substitution, suggesting a de novo genetic lesion. From these data it can be hypothesized that the Q167K mutation has a dominant negative effect.

Genetic polymorphisms of antioxidant enzymes in preterm infants

Background: Oxidative stress (OS) is significantly involved in the development of several complications associated with preterm birth, such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP) and intraventricular hemorrhage (IVH). Evidence is growing about the associations between single nucleotide polymorphisms (SNPs) in genes involved in OS or antioxidant response and the occurrence of neonatal morbidities. Aim of the study: To assess whether SNPs in genes of superoxide dismutase (SOD) and catalase (CAT), involved in antioxidant pathways, correlate with the occurrence of RDS, BPD, IVH and ROP in preterm neonates. Methods: We performed a retrospective study involving neonates <28 weeks of gestational age. Results: We demonstrated that rs8192287 SOD3 polymorphism is an independent protective factor for IVH, while rs4880 and rs5746136 SOD2 polymorphisms are associated with lower gestational age and birth weight. Haplotypes reconstruction showed that SOD1 (GG) decreased the risk of RDS, IVH and ROP; SOD2 (GT) increased the risk of BPD and decreased the risk of RDS, IVH, and ROP; SOD3 (TGC) decreased the risk of BPD and IVH; and CAT (CTC) decreased the risk of RDS. Conclusions: The study of SNPs or haplotypes reconstruction in genes involved in OS or scavenging activity may be helpful in identifying preterm newborns with a particularly high risk of morbidities, who may benefit from specific prevention strategies.

Neonatal onset of hyperornithinemia-hyperammonemia-homocitrullinuriasyndrome with favorable outcome

We report on a neonate with hyperammonemic coma in whom hyperornithinemia-hyperammonemia-homocitrullinuria syndrome was diagnosed. Appropriate treatment led to rapid clinical and metabolic improvement. The incorporation of 14C-ornithine on cultured fibroblasts confirmed the diagnosis. At the age of 18 months, the patient is in excellent clinical condition.