Serena Grossi

IDUA mutational profiling of a cohort of 102 European patients with mucopolysaccharidosis type I: identification and characterization of 35 novel α-L-iduronidase (IDUA) alleles

Mutational analysis of the IDUA gene was performed in a cohort of 102 European patients with mucopolysaccharidosis type I. A total of 54 distinct mutant IDUA alleles were identified, 34 of which were novel including 12 missense mutations, 2 nonsense mutations, 12 splicing mutations, 5 micro‐deletions, 1 micro‐duplication 1 translational initiation site mutation, and 1 ‘no‐stop’ change (p.X654RextX62). Evidence for the pathological significance of all novel mutations identified was sought by means of a range of methodological approaches, including the assessment of evolutionary conservation, RT‐PCR/in vitro splicing analysis, MutPred analysis and visual inspection of the 3D‐model of the IDUA protein. Taken together, these data not only demonstrate the remarkable mutational heterogeneity characterizing type 1 mucopolysaccharidosis but also illustrate our increasing ability to make deductions pertaining to the genotype‐phenotype relationship in disorders manifesting a high degree of allelic heterogeneity.

Biochemical and molecular findings in a patient with myoclonic epilepsy due to a mistarget of the β-glucosidase enzyme

A deficiency of human LIMP-2, a receptor for lysosomal mannose 6-phosphate-independent targeting of the beta-glucosidase (betaGC), due to mutations in the SCARB2 gene was described only in six families presented with progressive myoclonic epilepsy and nephrotic syndrome. In one of them a mistarget of the betaGC was demonstrated. We report here the biochemical and molecular findings in a patient diagnosed with progressive myoclonic epilepsy due to a mistarget of the betaGC, probably caused by a LIMP-2 deficiency, providing valuable information for the diagnosis of this rare disorder.

Molecular Genetic Analysis of the PLP1 Gene in 38 Families with PLP1-related disorders: Identification and Functional Characterization of 11 Novel PLP1 Mutations

BackgroundThe breadth of the clinical spectrum underlying Pelizaeus-Merzbacher disease and spastic paraplegia type 2 is due to the extensive allelic heterogeneity in the X-linked PLP1 gene encoding myelin proteolipid protein (PLP). PLP1 mutations range from gene duplications of variable size found in 60-70% of patients to intragenic lesions present in 15-20% of patients.MethodsForty-eight male patients from 38 unrelated families with a PLP1-related disorder were studied. All DNA samples were screened for PLP1 gene duplications using real-time PCR. PLP1 gene sequencing analysis was performed on patients negative for the duplication. The mutational status of all 14 potential carrier mothers of the familial PLP1 gene mutation was determined as well as 15/24 potential carrier mothers of the PLP1 duplication.Results and ConclusionsPLP1 gene duplications were identified in 24 of the unrelated patients whereas a variety of intragenic PLP1 mutations were found in the remaining 14 patients. Of the 14 different intragenic lesions, 11 were novel; these included one nonsense and 7 missense mutations, a 657-bp deletion, a microdeletion and a microduplication. The functional significance of the novel PLP1 missense mutations, all occurring at evolutionarily conserved residues, was analysed by the MutPred tool whereas their potential effect on splicing was ascertained using the Skippy algorithm and a neural network. Although MutPred predicted that all 7 novel missense mutations would be likely to be deleterious, in silico analysis indicated that four of them (p.Leu146Val, p.Leu159Pro, p.Thr230Ile, p.Ala247Asp) might cause exon skipping by altering exonic splicing elements. These predictions were then investigated in vitro for both p.Leu146Val and p.Thr230Ile by means of RNA or minigene studies and were subsequently confirmed in the case of p.Leu146Val. Peripheral neuropathy was noted in four patients harbouring intragenic mutations that altered RNA processing, but was absent from all PLP1-duplication patients. Unprecedentedly, family studies revealed the de novo occurrence of the PLP1 duplication at a frequency of 20%.

PLP1 gene duplication causes overexpression and alteration of the PLP/DM20 splicing balance in fibroblasts from Pelizaeus-Merzbacher disease patients

The PLP1 gene encodes two protein isoforms (PLP and DM20) which represent the predominant protein portion in myelin of the central nervous system. The two products are generated from the same primary transcript by alternative splicing. Defects of the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) or X-linked spastic paraplegia type 2 (SPG2). Duplication of the PLP1 gene is the most frequent gene defect, usually responsible for the classic form of PMD. To investigate the effects of PLP1 gene over dosage on gene expression, we analysed the PLP/DM20 expression profile in fibroblasts from three PMD patients with a PLP1 gene duplication. Gene expression was evaluated by real-time PCR using two different PLP1 amplicons and two different reference genes (GAPDH and GUSB). Fibroblasts from the three patients showed a 4-5 fold increase of PLP1 gene expression compared to fibroblasts from three normal controls. The contribution of the two alternatively spliced transcript isoforms (PLP and DM20) to the whole PLP1 gene expression was investigated using a DM20-specific amplicon. The three patients showed a decrease of the DM20/(DM20+PLP) ratio in comparison to the three normal controls, suggesting a prominent contribution of the PLP transcript to the PLP1 gene overexpression detected in the patients. Therefore, PLP1 gene duplication seems to result both in overexpression and in a shift of the PLP/DM20 splicing balance in direction of the PLP isoform.

Molecular analysis of ARSA and PSAP genes in twenty-one Italian patients with metachromatic leukodystrophy: identification and functional characterization of 11 novel ARSA alleles.

Metachromatic leukodystrophy (MLD), the demyelinating disorder resulting from impaired sulfatide catabolism, is caused by allelic mutations of the Arylsulfatase A (ARSA) locus except for extremely rare cases of Saposin‐B (Sap‐B) deficiency. We characterized twenty‐one unrelated Italian patients among which seventeen were due to ARSA activity deficiency and 4 others resulted from Saposin‐B defect. Overall, we found 20 different mutant ARSA alleles and 2 different Sap‐B alleles. The eleven new ARSA alleles (c.53C>A; c.88G>C; c.372G>A; c.409_411delCCC; c.634G>C; [c.650G>A;c.1108C>T]; c.845A>G; c.906G>C; c.919G>T; c.1102‐3C>G; c.1126T>A) were functionally characterized and the novel amino acid changes were also modelled into the three‐dimensional structure. The present study is aimed at providing a broader picture of the molecular basis of MLD in the Italian population. It also emphasizes the importance of a comprehensive evaluation in MLD diagnosis including biochemical, enzymatic and molecular investigations.

Diagnosis of Pelizaeus–Merzbacher disease: detection of proteolipid protein gene copy number by real-time PCR

Duplication of the proteolipid protein gene (PLP1) is the most frequent cause of Pelizaeus–Merzbacher disease (PMD), a severe X-linked myelination disorder. We developed an assay for the detection of the PLP1 gene dosage by real-time quantitative PCR using the ABI Prism 7700 Sequence Detection System and the TaqMan chemistry. Copy number of the PLP1 gene was determined by the standard curve method using GAPDH as the reference gene. The assay was tested both on 50 normal controls and on 20 subjects whose PLP1 gene copy number was previously determined by quantitative fluorescent multiplex PCR. The procedure confirmed the expected results both on the male and female normal controls as well as on the 20 subjects previously tested. Ratios corresponding to the presence of one, two or three PLP1 gene copies, distributed in three non-overlapping ranges, were obtained by real-time PCR analysis. Subsequently, 29 DNA samples of putative PMD patients and possible female carriers, with unknown PLP1 gene dosage, were analysed. Five affected males carrying the PLP1 gene duplication and four female heterozygotes carrying three PLP1 gene copies were identified among them. The method is suitable for the identification of affected male patients and female carriers. Specific ranges are widely spaced, ensuring a correct assignment of the PLP1 gene copy number.

Functional analysis of 11 novel GBA alleles

Gaucher disease is the most frequent lysosomal storage disorder due to the deficiency of the acid β-glucosidase, encoded by the GBA gene. In this study, we report the structural and functional characterization of 11 novel GBA alleles. Seven single missense alleles, P159S, N188I, E235K, P245T, W312S, S366R and W381C, and two alleles carrying in cis mutations, (N188S; G265R) and (E326K; D380N), were studied for enzyme activity in transiently transfected cells. All mutants were inactive except the P159S, which retained 15% of wild-type activity. To further characterize the alleles carrying two in cis mutations, we expressed constructs bearing singly each mutation. The presence of G265R or D380N mutations completely abolished enzyme activity, while N188S and E326K mutants retained 25 and 54% of wild-type activity, respectively. Two mutations, affecting the acceptor splice site of introns 5 (c.589-1G>A) and 9 (c.1389-1G>A), led to the synthesis of aberrant mRNA. Unpredictably, family studies showed that two alleles resulted from germline or ‘de novo’ mutations. These results strengthen the importance of performing a complete and accurate molecular analysis of the GBA gene in order to avoid misleading conclusions and provide a comprehensive functional analysis of new GBA mutations.

Early Visual Seizures and Progressive Myoclonus Epilepsy in Neuronopathic Gaucher Disease Due to a Rare Compound Heterozygosity (N188S/S107L)

Summary:  Purpose: Gaucher disease, the inherited deficiency of the lysosomal enzyme glucocerebrosidase, is characterized by genotypic and phenotypic heterogeneity. We recently characterized the glucocerebrosidase alleles of a patient with an unusual clinical presentation of type 3 Gaucher disease.

Mutation Update of ARSA and PSAP Genes Causing Metachromatic Leukodystrophy

Metachromatic leukodystrophy is a neurodegenerative disorder characterized by progressive demyelination. The disease is caused by variants in the ARSA gene, which codes for the lysosomal enzyme arylsulfatase A, or, more rarely, in the PSAP gene, which codes for the activator protein saposin B. In this Mutation Update, an extensive review of all the ARSA‐ and PSAP‐causative variants published in the literature to date, accounting for a total of 200 ARSA and 10 PSAP allele types, is presented. The detailed ARSA and PSAP variant lists are freely available on the Leiden Online Variation Database (LOVD) platform at http://www.LOVD.nl/ARSA and http://www.LOVD.nl/PSAP, respectively.

Haplotype analysis suggests a single Balkan origin for the Gaucher disease [D409H;H255Q] double mutant allele

Gaucher disease is an autosomal recessive lysosomal storage disease that is mainly due to mutations in the GBA gene. Most of the mutant alleles described so far bear a single mutation. However, there are a few alleles bearing two or more DNA changes. It has been reported that patients homozygous for the [D409H;H255Q] double mutant allele (HGVS‐approved nomenclature, p.[D448H;H294Q]) present a more severe phenotype than patients homozygous for the relatively common D409H mutation. In this study, we confirmed the detrimental cumulative effect of these two mutations at the enzymatic activity level by the heterologous expression of the single and double mutant alleles. Additionally, we found a high frequency of the [D409H;H255Q] allele in patients from the Balkans and the Adriatic area of Italy. This prompted us to perform a haplotype analysis, using five microsatellite polymorphisms close to the GBA gene, to determine the origin of this allele. The results of the 37 chromosomes analysed showed that most of them share a common haplotype and are consistent with a single origin in the Balkans and the Adriatic area of Italy for the [D409H;H255Q] allele.