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

AbstractMucopolysaccharidosis 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 ΔS117) 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

Further genotype-phenotype correlation emerging from two families with PLP1 exon 4 skipping

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Cell Line and DNA Biobank From Patients Affected by Genetic Diseases

The Bioresource, presently storing 10,279 biospecimens, was initially established in 1976 as a private laboratory-collection to maintain rare mutant cell lines from genetic-metabolic diseases. Shortly afterwards, however, data from the sample collection was organised in a database and the sample collection was released to the scientific community. The Biobank has received Telethon grants since 1993, as individual facility, and from 2008 as part of the Telethon Network of Genetic Biobanks (www.biobanknetwork.org).In 2010, the Biobank has obtained official recognition from Regione Liguria. The Biobank has always provided essential services by establishing, analysing, maintaining, and distributing biospecimens from patients affected by rare genetic diseases. Up to now, the contribution of the Biobank to the scientific community has been expressed in acknowledgement notes in 145 scientific manuscripts

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials

In vitro recapitulation of the site-specific editing (to wild-type) of mutant IDS mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs

The transfer of genomic information into the primary RNA sequence can be altered by RNA editing. We have previously shown that genomic variants can be RNA-edited to wild-type. The presence of distinct “edited” iduronate 2-sulfatase (IDS) mRNA transcripts ex vivo evidenced the correction of a nonsense and frameshift variant, respectively, in three unrelated Hunter syndrome patients. This phenomenon was confirmed in various patient samples by a variety of techniques, and was quantified by single-nucleotide primer extension. Western blotting also confirmed the presence of IDS protein similar in size to the wild-type. Since preliminary experimental evidence suggested that the “corrected” IDS proteins produced by the patients were similar in molecular weight and net charge to their wild-type counterparts, an in vitro system employing different cell types was established to recapitulate the site-specific editing of IDS RNA (uridine to cytidine conversion and uridine deletion), and to confirm the findings previously observed ex vivo in the three patients. In addition, confocal microscopy and flow cytometry analyses demonstrated the expression and lysosomal localization in HEK293 cells of GFP-labeled proteins translated from edited IDS mRNAs. Confocal high-content analysis of the two patients’ cells expressing wild-type or mutated IDS confirmed lysosomal localization and showed no accumulation in the Golgi or early endosomes

Identification and Characterization of 15 Novel GALC Gene Mutations Causing Krabbe Disease

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region. © 2010 Wiley-Liss, Inc

A self-repair history: compensatory effect of a de novo variant on the FANCA c.2778+83C>G splicing mutation

Introduction: Fanconi anemia (FA) is a genome instability condition that drives somatic mosaicism in up to 25% of all patients, a phenomenon now acknowledged as a good prognostic factor. Herein, we describe the case of P1, a FA proband carrying a splicing variant, molecularly compensated by a de novo insertion. Methods and Results: Targeted next-generation sequencing on P1's peripheral blood DNA detected the known FANCA c.2778 + 83C > G intronic mutation and suggested the presence of a large deletion on the other allele, which was then assessed by MLPA and RT-PCR. To determine the c.2778 + 83C > G splicing effect, we performed a RT-PCR on P1's lymphoblastoid cell line (LCL) and on the LCL of another patient (P2) carrying the same variant. Although we confirmed the expected alternative spliced form with a partial intronic retention in P2, we detected no aberrant products in P1's sample. Sequencing of P1's LCL DNA allowed identification of the de novo c.2778 + 86insT variant, predicted to compensate 2778 + 83C > G impact. Albeit not found in P1's bone marrow (BM) DNA, c.2778 + 86insT was detected in a second P1's LCL established afterward, suggesting its occurrence at a low level in vivo. Minigene assay recapitulated the c.2778 + 83C > G effect on splicing and the compensatory role of c.2778 + 86insT in re-establishing the physiological mechanism. Accordingly, P1's LCL under mitomycin C selection preserved the FA pathway activity in terms of FANCD2 monoubiquitination and cell survival. Discussion: Our findings prove the role of c.2778 + 86insT as a second-site variant capable of rescuing c.2778 + 83C > G pathogenicity in vitro, which might contribute to a slow hematopoietic deterioration and a mild hematologic evolution

Hypomorphic FANCA mutations correlate with mild mitochondrial and clinical phenotype in Fanconi anemia

Fanconi anemia is a rare disease characterized by congenital malformations, aplastic anemia, and predisposition to cancer. Despite the consolidated role of the Fanconi anemia proteins in DNA repair, their involvement in mitochondrial function is emerging. The purpose of this work was to assess whether the mitochondrial phenotype, independent of genomic integrity, could correlate with patient phenotype. We evaluated mitochondrial and clinical features of 11 affected individuals homozygous or compound heterozygous for p.His913Pro and p.Arg951Gln/Trp, the two residues of FANCA that are more frequently affected in our cohort of patients. Although p.His913Pro and p.Arg951Gln proteins are stably expressed in cytoplasm, they are unable to migrate in the nucleus, preventing cells from repairing DNA. In these cells, the electron transfer between respiring complex I-III is reduced and the ATP/AMP ratio is impaired with defective ATP production and AMP accumulation. These activities are intermediate between those observed in wild-type and FANCA-/- cells, suggesting that the variants at residues His913 and Arg951 are hypomorphic mutations. Consistent with these findings, the clinical phenotype of most of the patients carrying these mutations is mild. These data further support the recent finding that the Fanconi anemia proteins play a role in mitochondria, and open up possibilities for genotype/phenotype studies based on novel mitochondrial criteria

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

<p>Abstract</p> <p>Background</p> <p>The 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 <it>PLP1 </it>gene encoding myelin proteolipid protein (PLP). <it>PLP1 </it>mutations range from gene duplications of variable size found in 60-70% of patients to intragenic lesions present in 15-20% of patients.</p> <p>Methods</p> <p>Forty-eight male patients from 38 unrelated families with a PLP1-related disorder were studied. All DNA samples were screened for <it>PLP1 </it>gene duplications using real-time PCR. <it>PLP1 </it>gene sequencing analysis was performed on patients negative for the duplication. The mutational status of all 14 potential carrier mothers of the familial <it>PLP1 </it>gene mutation was determined as well as 15/24 potential carrier mothers of the <it>PLP1 </it>duplication.</p> <p>Results and Conclusions</p> <p><it>PLP1 </it>gene duplications were identified in 24 of the unrelated patients whereas a variety of intragenic <it>PLP1 </it>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 <it>PLP1 </it>missense mutations, all occurring at evolutionarily conserved residues, was analysed by the <it>MutPred </it>tool whereas their potential effect on splicing was ascertained using the <it>Skippy </it>algorithm and a neural network. Although <it>MutPred </it>predicted that all 7 novel missense mutations would be likely to be deleterious, <it>in silico </it>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 <it>in vitro </it>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 <it>PLP1</it>-duplication patients. Unprecedentedly, family studies revealed the <it>de novo </it>occurrence of the <it>PLP1 </it>duplication at a frequency of 20%.</p

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology

Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes