MLPA-based approach for initial and simultaneous detection of GBA deletions and recombinant alleles in patients affected by Gaucher Disease.

The chromosomal region, in which the GBA gene is located, is structurally subject to misalignments, reciprocal and nonreciprocal homologous recombination events, leading to structural defects such as deletions, duplications and gene-pseudogene complex rearrangements causing Gaucher Disease (GD). Interestingly deletions and duplications, belonging to the heterogeneous group of structural defects collectively termed Copy Number Variations (CNVs), together with gene-pseudogene complex rearrangements represent the main cause of pitfalls in GD mutational analysis. In the present study, we set up and validate a Multiplex Ligation-dependent Probe Amplification (MLPA)-based approach to simultaneously investigate the potential occurrence of CNVs and complex rearrangements in 8 unrelated GD patients who had still not-well-characterized or uncharacterized alleles. The findings allowed us to complete the mutational analysis in 4 patients, identifying a rare deletion (g.-3100_+834del3934) and 2 novel recombinant alleles (g.4356_7031conJ03060.1:g.2544_4568; g.1942_7319conJ03060.1:g.1092_4856). These results demonstrate the diagnostic usefulness of MLPA in the detection of GBA deletions and recombinations. In addition, MLPA findings have also served as a basis for developing molecular approaches to precisely pinpoint the breakpoints and characterize the underlying mechanism of copy number variations.

A novel homozygous splicing mutation in PSAP gene causes metachromatic leukodystrophy in two Moroccan brothers.

Prosaposin (PSAP) gene mutations, affecting saposin B (Sap-B) domain, cause a rare metachromatic leukodystrophy (MLD) variant in which arylsulfatase A (ARSA) activity is normal. To date, only 10 different PSAP mutations have been associated with a total of 18 unrelated MLD patients worldwide. In this study, we report for the first time a family with Moroccan origins in which the proband, presenting with a late-infantile onset of neurological involvement and a brain MRI with the typical tigroid MLD pattern, showed normal values of ARSA activity in the presence of an abnormal pattern of urinary sulfatides. In view of these findings, PSAP gene was analyzed, identifying the newly genomic homozygous c.909 + 1G > A mutation occurring within the invariant GT dinucleotide of the intron 8 donor splice site. Reverse transcriptase-polymerase chain reaction (RT-PCR), showing the direct junction of exon 7 to exon 9, confirmed the skipping of the entire exon 8 (p.Gln260_Lys303) which normally contains two cysteine residues (Cys271 and Cys265) involved in disulfide bridges. Our report provides further evidence that phenotypes of patients with Sap-B deficiency vary widely depending on age of onset, type, and severity of symptoms. Awareness of this rare MLD variant is crucial to prevent delayed diagnosis or misdiagnosis and to promptly provide an accurate genetic counseling, including prenatal diagnosis, to families.

ASAH1 variant causing a mild SMA phenotype with no myoclonic epilepsy: a clinical, biochemical and molecular study.

ASAH1 gene encodes for acid ceramidase that is involved in the degradation of ceramide into sphingosine and free fatty acids within lysosomes. ASAH1 variants cause both the severe and early-onset Farber disease and rare cases of spinal muscular atrophy (SMA) with progressive myoclonic epilepsy (SMA-PME), phenotypically characterized by childhood onset of proximal muscle weakness and atrophy due to spinal motor neuron degeneration followed by occurrence of severe and intractable myoclonic seizures and death in the teenage years. We studied two subjects, a 30-year-old pregnant woman and her 17-year-old sister, affected with a very slowly progressive non-5q SMA since childhood. No history of seizures or myoclonus has been reported and EEG was unremarkable. The molecular study of ASAH1 gene showed the presence of the homozygote nucleotide variation c.124A>G (r.124a>g) that causes the amino acid substitution p.Thr42Ala. Biochemical evaluation of cultured fibroblasts showed both reduction in ceramidase activity and accumulation of ceramide compared with the normal control. This study describes for the first time the association between ASAH1 variants and an adult SMA phenotype with no myoclonic epilepsy nor death in early age, thus expanding the phenotypic spectrum of ASAH1-related SMA. ASAH1 molecular analysis should be considered in the diagnostic testing of non-5q adult SMA patients.

The alliance between genetic biobanks and patient organisations: the experience of the telethon network of genetic biobanks.

BACKGROUND: Rare diseases (RDs) are often neglected because they affect a small percentage of the population (6-8 %), which makes research and development of new therapies challenging processes. Easy access to high-quality samples and associated clinical data is therefore a key prerequisite for biomedical research. In this context, Genetic Biobanks are critical to developing basic, translational and clinical research on RDs. The Telethon Network of Genetic Biobanks (TNGB) is aware of the importance of biobanking as a service for patients and has started a dialogue with RD-Patient Organisations via promotion of dedicated meetings and round-tables, as well as by including their representatives on the TNGB Advisory Board. This has enabled the active involvement of POs in drafting biobank policies and procedures, including those concerning ethical issues. Here, we report on our experience with RD-Patient Organisations who have requested the services of existing biobanks belonging to TNGB and describe how these relationships were established, formalised and maintained. RESULTS: The process of patient engagement has proven to be successful both for lay members, who increased their understanding of the complex processes of biobanking, and for professionals, who gained awareness of the needs and expectations of the people involved. This collaboration has resulted in a real interest on the part of Patient Organisations in the biobanking service, which has led to 13 written agreements designed to formalise this process. These agreements enabled the centralisation of rare genetic disease biospecimens and their related data, thus making them available to the scientific community. CONCLUSIONS: The TNGB experience has proven to be an example of good practice with regard to patient engagement in biobanking and may serve as a model of collaboration between disease-oriented Biobanks and Patient Organisations. Such collaboration serves to enhance awareness and trust and to encourage the scientific community to address research on RDs.

Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1.

Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to an autosomal recessive deficiency of acid beta-glucosidase characterized by the accumulation of glucocerebroside. In this work we carried out the molecular analysis of the glucocerebrosidase gene (GBA) in 58 unrelated patients with GD type 1. We identified five novel genetic alterations: three missense changes c.187G>A (p.D63N), c.473T>G (p.I158S), c.689T>A (p.V230E), a gene-pseudogene recombinant allele and a non-pseudogene-derived complex allele [c.1379G>A;c.1469A>G] encoding [p.G460D;p.H490R]. All mutant alleles were present as compound heterozygotes in association with c.1226A>G (p.N409S), the most common mutation in GD1. The missense mutant proteins were expressed in vitro in COS-1 cells and analyzed by enzyme activity, protein processing and intracellular localization. Functional studies also included the c.662C>T (p.P221L) mutation recently reported in the Spanish GD population (Montfort et al., 2004). The missense mutant alleles retained an extremely low residual enzyme activity with respect to wild type; the complex allele expressed no activity. Processing of the mutant proteins was unaltered except for c.473T>G which was differently glycosylated due to the exposition of an additional glycosylation site. Immunofluorescence studies showed that protein trafficking into the lysosomes was unaffected in all cases. Finally, the characterization of the novel recombinant allele identified a crossover involving the GBA gene and pseudogene between intron 5 and exon 7.

Analysis of the glucocerebrosidase gene and mutation profile in 144 Italian gaucher patients.

Gaucher disease (GD), the most prevalent lysosomal storage disease characterized by a remarkable degree of clinical variability, results from deleterious mutations in the glucocerebrosidase gene (GBA). In this paper we report the molecular characterization of 144 unrelated Italian GD patients with the three types of the disease. The allelic frequencies of Italians are reported and the mutation profile is analyzed. Besides the common N370S, L444P, RecNciI, G202R, IVS2+1G>A, D409H, F213I mutations, the different molecular strategies, used for the mutation detection, identified the rare N107L, R131C, R170C, R170P, N188S, S196P, R285C, R285H, W312C, D399N, A446P, IVS10-1G>A, RecDelta55, total gene deletion, as well as 12 mutant alleles that were exclusively present in the Italian population until now: the previously reported R353G, N370S+S488P mosaicism, IVS8(-11delC)-14T>A), Rec I, Y418C, and the seven novel alleles D127X, P159T, V214X, T231R, L354X, H451R, and G202R+M361I. The wide phenotypic differences observed within the genotypic groups as well as between siblings implicate a significant contribution of other modifying genetic and/or non-genetic factors and claim a comprehensive valuation of the patient including clinical., biochemical and molecular investigations for prognosis, appropriate interventive therapy and reliable genetic counseling.

Early visual seizures and progressive myoclonus epilepsy in neuronopathic Gaucher disease due to a rare compound heterozygosity (N188S/S107L).

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. METHODS: Initial clinical manifestations appeared at age 11 years as visual seizures. RESULTS: Subsequent progressive myoclonus and generalized seizures were consistent with an adolescent-onset form of progressive myoclonus epilepsy. However, a specific diagnosis was established only at age 16, because of the absence of hematologic abnormalities and a fairly moderate hepatomegaly. Bone marrow aspirate was slightly positive for Gaucher cells. Demonstration of reduced glucocerebrosidase in the fibroblasts confirmed the diagnosis. The child died at age 19 years. Postmortem sequencing of the glucocerebrosidase gene from cultured fibroblasts demonstrated a rare compound heterozygote for N188S/S107L. CONCLUSIONS: This unusual presentation of Gaucher disease indicates that if clinical and neurophysiological findings in adolescents with initial visual seizures and myoclonus suggest a progressive disorder, enzymatic assay is mandatory, even in the absence of the classic neurologic and systemic signs of the disease. Early differential diagnosis from other forms of progressive myoclonus epilepsy with similar clinical presentation may help provide appropriate genetic counseling.