Giulia Soldà

Survival and dementia in GBA-associated Parkinson's disease: The mutation matters.

The objective of this work was to investigate survival, dementia, and genotype‐phenotype correlations in patients with Parkinsons disease (PD) with and without mutations on the glucocerebrosidase gene (GBA).

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

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.

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

In-depth characterization of breast cancer tumor-promoting cell transcriptome by RNA sequencing and microarrays

Numerous studies have reported the existence of tumor-promoting cells (TPC) with self-renewal potential and a relevant role in drug resistance. However, pathways and modifications involved in the maintenance of such tumor subpopulations are still only partially understood. Sequencing-based approaches offer the opportunity for a detailed study of TPC including their transcriptome modulation. Using microarrays and RNA sequencing approaches, we compared the transcriptional profiles of parental MCF7 breast cancer cells with MCF7-derived TPC (i.e. MCFS). Data were explored using different bioinformatic approaches, and major findings were experimentally validated. The different analytical pipelines (Lifescope and Cufflinks based) yielded similar although not identical results. RNA sequencing data partially overlapped microarray results and displayed a higher dynamic range, although overall the two approaches concordantly predicted pathway modifications. Several biological functions were altered in TPC, ranging from production of inflammatory cytokines (i.e., IL-8 and MCP-1) to proliferation and response to steroid hormones. More than 300 non-coding RNAs were defined as differentially expressed, and 2,471 potential splicing events were identified. A consensus signature of genes up-regulated in TPC was derived and was found to be significantly associated with insensitivity to fulvestrant in a public breast cancer patient dataset. Overall, we obtained a detailed portrait of the transcriptome of a breast cancer TPC line, highlighted the role of non-coding RNAs and differential splicing, and identified a gene signature with a potential as a context-specific biomarker in patients receiving endocrine treatment.

Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis

Alport syndrome (AS) is an inherited progressive renal disease caused by mutations in COL4A3, COL4A4, and COL4A5 genes. Despite simultaneous screening of these genes being widely available, mutation detection still remains incomplete in a non-marginal portion of patients. Here, we applied whole-exome sequencing (WES) in 3 Italian families negative after candidate-gene analyses. In Family 1, we identified a novel heterozygous intronic variant (c.2245-40A>G) -outside the conventionally screened candidate region for diagnosis- potentially disrupting COL4A5 exon29 splicing. Using a minigene-based approach in HEK293 cells we demonstrated that this variant abolishes exon29 branch site, causing exon skipping. Moreover, skewed X-inactivation of the c.2245-40A>G allele correlated with disease severity in heterozygous females. In Family 2, WES highlighted a novel COL4A5 hemizygous missense mutation (p.Gly491Asp), which segregates with the phenotype and impacts on a highly-conserved residue. Finally, in Family 3, we detected a homozygous 24-bp in-frame deletion in COL4A3 exon1 (NM_000091.4:c.30_53del:p.Val11_Leu18del or c.40_63del24:p.Leu14_Leu21del), which is ambiguously annotated in databases, although it corresponds to a recurrent AS mutation. Functional analyses showed that this deletion disrupts COL4A3 signal peptide, possibly altering protein secretion. In conclusion, WES -together with functional studies- was fundamental for molecular diagnosis in 3 AS families, highlighting pathogenic variants that escaped previous screenings.