Letizia Straniero

DNAJC12 and dopa-responsive nonprogressive parkinsonism

Biallelic DNAJC12 mutations were described in children with hyperphenylalaninemia, neurodevelopmental delay, and dystonia. We identified DNAJC12 homozygous null variants (c.187A>T;p.K63* and c.79‐2A>G;p.V27Wfs*14) in two kindreds with early‐onset parkinsonism. Both probands had mild intellectual disability, mild nonprogressive, motor symptoms, sustained benefit from small dose of levodopa, and substantial worsening of symptoms after levodopa discontinuation. Neuropathology (Proband‐A) revealed no alpha‐synuclein pathology, and substantia nigra depigmentation with moderate cell loss. DNAJC12 transcripts were reduced in both patients. Our results suggest that DNAJC12 mutations (absent in 500 early‐onset patients with Parkinsons disease) rarely cause dopa‐responsive nonprogressive parkinsonism in adulthood, but broaden the clinical spectrum of DNAJC12 deficiency. Ann Neurol 2017;82:640–646

Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants.

Whole-gene CFTR sequencing combined with digital RT-PCR improves genetic diagnosis of cystic fibrosis

Despite extensive screening, 1–5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG[11–12]T5 allele—known to be associated with decreased CFTR mRNA levels—the molecular diagnosis was implemented by measuring the residual level of wild-type transcript by digital reverse transcription polymerase chain reaction performed on RNA extracted from nasal brushing.

The GBAP1 pseudogene acts as a ceRNA for the glucocerebrosidase gene GBA by sponging miR-22-3p.

Mutations in the GBA gene, encoding lysosomal glucocerebrosidase, represent the major predisposing factor for Parkinson’s disease (PD), and modulation of the glucocerebrosidase activity is an emerging PD therapy. However, little is known about mechanisms regulating GBA expression. We explored the existence of a regulatory network involving GBA, its expressed pseudogene GBAP1, and microRNAs. The high level of sequence identity between GBA and GBAP1 makes the pseudogene a promising competing-endogenous RNA (ceRNA), functioning as a microRNA sponge. After selecting microRNAs potentially targeting both transcripts, we demonstrated that miR-22-3p binds to and down-regulates GBA and GBAP1, and decreases their endogenous mRNA levels up to 70%. Moreover, over-expression of GBAP1 3′-untranslated region was able to sequester miR-22-3p, thus increasing GBA mRNA and glucocerebrosidase levels. The characterization of GBAP1 splicing identified multiple out-of-frame isoforms down-regulated by the nonsense-mediated mRNA decay, suggesting that GBAP1 levels and, accordingly, its ceRNA effect, are significantly modulated by this degradation process. Using skin-derived induced pluripotent stem cells of PD patients with GBA mutations and controls, we observed a significant GBA up-regulation during dopaminergic differentiation, paralleled by down-regulation of miR-22-3p. Our results describe the first microRNA controlling GBA and suggest that the GBAP1 non-coding RNA functions as a GBA ceRNA.

Identification of a novel large deletion in a patient with severe factor V deficiency using an in-house F5 MLPA assay

Factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by mutations in the F5 gene. FV‐deficient patients in whom no mutation or only one mutation is found may harbour large gene rearrangements, which are not detected by conventional mutation screening strategies. The aim of this study was to develop and validate a multiplex ligation‐dependent probe amplification (MLPA) assay for the detection of large deletions and duplications in the F5 gene. Twenty‐two MLPA probes targeting 19 of the 25 exons and the upstream and downstream regions of the F5 gene were designed and tested in 10 normal controls, a patient with a known heterozygous deletion of F5 exons 1–7 (positive control) and 14 genetically unexplained FV‐deficient patients. MLPA results were confirmed by digital PCR on a QuantStudio™ 3D Digital PCR System. The F5‐specific probes yielded a reproducible peak profile in normal controls, correctly detected the known deletion in the positive control and suggested the presence of a novel deletion of exons 9–10 in a patient with undetectable FV levels and only one identified mutation. Follow‐up by chip‐based digital PCR, long‐range PCR and direct sequencing confirmed that this patient carried a heterozygous F5 deletion of 1823 bp extending from intron 8 to intron 10. Bioinformatics sequence analysis pinpointed repetitive elements that might have originated the deletion. In conclusion, we have developed and validated an MLPA assay for the detection of gross F5 gene rearrangements. This assay may represent a valuable tool for the molecular diagnosis of FV deficiency.

A lysosome–plasma membrane–sphingolipid axis linking lysosomal storage to cell growth arrest

Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose‐loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids (i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp‐1 on the PM of sucrose‐loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose‐loaded fibroblasts. The inhibition of β‐ glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.—Samarani, M., Loberto, N., Soldà, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome‐plasma membrane‐sphingolipid axis linking lysosomal storage to cell growth arrest. FASEB J. 32, 5685–5702 (2018). www.fasebj.org

Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson’s disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

2,6-Difluorobenzamide Inhibitors of Bacterial Cell Division Protein FtsZ: Design, Synthesis, and Structure-Activity Relationships

A wide variety of drug‐resistant microorganisms are continuously emerging, restricting the therapeutic options for common bacterial infections. Antimicrobial agents that were originally potent are now no longer helpful, due to their weak or null activity toward these antibiotic‐resistant bacteria. In addition, none of the recently approved antibiotics affect innovative targets, resulting in a need for novel drugs with innovative antibacterial mechanisms of action. The essential cell division protein filamentous temperature‐sensitiveu2005Z (FtsZ) has emerged as a possible target, thanks to its ubiquitous expression and its homology to eukaryotic β‐tubulin. In the latest years, several compounds were shown to interact with this prokaryotic protein and selectively inhibit bacterial cell division. Recently, our research group developed interesting derivatives displaying good antibacterial activities against methicillin‐resistant Staphylococcus aureus, as well as vancomycin‐resistant Enterococcus faecalis and Mycobacterium tuberculosis. The aim of the present study was to summarize the structure–activity relationships of differently substituted heterocycles, linked by a methylenoxy bridge to the 2,6‐difluorobenzamide, and to validate FtsZ as the real target of this class of antimicrobials.