Anna Bartoletti-Stella

The genetic and metabolic signature of oncocytic transformation implicates HIF1α destabilization

We previously showed that disruptive complex I mutations in mitochondrial DNA are the main genetic hallmark of oncocytic tumors of the thyroid and kidney. We here report a high frequency of homoplasmic disruptive mutations in a large panel of oncocytic pituitary and head-and-neck tumors. The presence of such mutations implicates disassembly of respiratory complex I in vivo which in turn contributes to the inability of oncocytic tumors to stabilize HIF1alpha and to display pseudo-hypoxia. By utilizing transmitochondrial cytoplasmic hybrids (cybrids), we induced the shift to homoplasmy of a truncating mutation in the mitochondria-coded MTND1 gene. Such shift is associated with a profound metabolic impairment leading to the imbalance of alpha-ketoglutarate and succinate, the Krebs cycle metabolites which are the main responsible for HIF1alpha stabilization. We conclude that the main hallmarks of oncocytic transformation, namely the occurrence of homoplasmic disruptive mutations and complex I disassembly, may explain the benign nature of oncocytic neoplasms through lack of HIF1alpha stabilization.

A mutation in PAK3 with a dual molecular effect deregulates the RAS/MAPK pathway and drives an X-linked syndromic phenotype

Loss-of-function mutations in PAK3 contribute to non-syndromic X-linked intellectual disability (NS-XLID) by affecting dendritic spine density and morphology. Linkage analysis in a three-generation family with affected males showing ID, agenesis of corpus callosum, cerebellar hypoplasia, microcephaly and ichthyosis, revealed a candidate disease locus in Xq21.33q24 encompassing over 280 genes. Subsequent to sequencing all coding exons of the X chromosome, we identified a single novel variant within the linkage region, affecting a conserved codon of PAK3. Biochemical studies showed that, similar to previous NS-XLID-associated lesions, the predicted amino acid substitution (Lys389Asn) abolished the kinase activity of PAK3. In addition, the introduced residue conferred a dominant-negative function to the protein that drives the syndromic phenotype. Using a combination of in vitro and in vivo studies in zebrafish embryos, we show that PAK3(N389) escapes its physiologic degradation and is able to perturb MAPK signaling via an uncontrolled kinase-independent function, which in turn leads to alterations of cerebral and craniofacial structures in vivo. Our data expand the spectrum of phenotypes associated with PAK3 mutations, characterize a novel mechanism resulting in a dual molecular effect of the same mutation with a complex PAK3 functional deregulation and provide evidence for a direct functional impact of aberrant PAK3 function on MAPK signaling.

Messenger RNA processing is altered in autosomal dominant leukodystrophy

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.

Mitochondrial DNA mutations in oncocytic adnexal lacrimal glands of the conjunctiva.

O ncocytic neoplasms are characterized by a marked mitochondrial hyperplasia. They are of epithelial derivation and usually occur in endocrine and exocrine organs such as the thyroid, parathyroids, kidney, pituitary, and salivary glands. Oncocytic lesions of the ophthalmic regions have been reported, especially as oncocytic adenomas of the caruncle. Similar lesions occur rarely in the adnexal lacrimal glands of the conjunctiva. Truncating mitochondrial DNA (mtDNA) mutations in respiratory complex I (CI) subunits have been suggested to be the genetic hallmark of the oncocytic phenotype. The occurrence of such genetic lesions strongly impairs cell respiration and contributes to maintaining the tumor in a low-proliferating, benign state. We describe an oncocytoma of the adnexal lacrimal glands of the conjunctiva harboring mtDNA mutations that cause CI disassembly and associate with a low proliferation index.

Muscle ceroid lipofuscin‐like deposits in a patient with corticobasal syndrome due to a progranulin mutation

details refer to online supporting data). Consequently, mutant huntingtin could have directly or indirectly triggered a progressive loss of type II fibers and a concomitant gain in type I fibers, as previously suggested by Strand and colleagues. In previous studies, there is evidence that mutant huntingtin affects neuronal mitochondrial function and thereby inhibits oxidative phosphorylation. In analogy, the higher type I fiber distribution in HD patients could represent a compensation for the lower respiratory capacity of complex I and oxidative phosphorylation capacity. HD is associated with altered vastus lateralis muscle fiber type distribution and changes in mitochondrial function, potentially supporting mitochondrial dysfunction to be a pathogenic factor in HD. Hence, the present data supports previous findings suggesting skeletal muscle tissue as a potential marker of disease progression in HD patients. Nonetheless, further studies investigating the underlying mechanisms are required.

Identification of rare genetic variants in Italian patients with dementia by targeted gene sequencing

Genetics is intricately involved in the etiology of neurodegenerative dementias. The incidence of monogenic dementia among all neurodegenerative forms is unknown due to the lack of systematic studies and of patient/clinician access to extensive diagnostic procedures. In this study, we conducted targeted sequencing in 246 clinically heterogeneous patients, mainly with early-onset and/or familial neurodegenerative dementia, using a custom-designed next-generation sequencing panel covering 27 genes known to harbor mutations that can cause different types of dementia, in addition to the detection of C9orf72 repeat expansions. Forty-nine patients (19.9%) carried known pathogenic or novel, likely pathogenic, variants, involving both common (presenilin 1, presenilin 2, C9orf72, and granulin) and rare (optineurin, serpin family I member 1 and protein kinase cyclic adenosine monophosphate (cAMP)-dependent type I regulatory subunit beta) dementia-associated genes. Our results support the use of an extended next-generation sequencing panels as a quick, accurate, and cost-effective method for diagnosis in clinical practice. This approach could have a significant impact on the proportion of tested patients, especially among those with an early disease onset.

Skin biopsy and microneurography disclose selective noradrenergic dysfunction due to dopamine-β-hydroxylase deficiency

Skin biopsy and microneurography are autonomic tests directly evaluating adrenergic and cholinergic sympathetic fibers to identify selective deficiency of a specific peripheral sympathetic subdivision. We describe a patient with tomacular neuropathy due to a deletion of the PMP22 gene who complained of chronic orthostatic hypotension due to a dopamine-β-hydroxylase deficiency confirmed by genetic analysis demonstrating two novel mutations in the DβH gene. To further characterize autonomic dysfunctions the proband underwent skin biopsy and microneurography. These tests disclosed a selective peripheral adrenergic dysfunction demonstrating the possibility to ascertain DβH deficiency. In conclusion, skin biopsy and microneurography may help to increase the diagnosis of this peculiar disorder particularly when routine autonomic nervous system tests show uncertain results.

Age at onset of genetic (E200K) and sporadic Creutzfeldt-Jakob diseases is modulated by the CYP4X1 gene

Objectives The Glu to Lys change at codon 200 (E200K) of the PRNP gene is the most frequent mutation associated to genetic Creutzfeldt-Jakob disease (CJD) and the only one responsible for geographical clusters. Patients carrying this mutation develop disease at different ages and show variable clinical phenotypes that are not affected by the methione/valine polymorphism at codon 129 of the PRNP gene suggesting the influence of other factors. The objective of this study is to look for genes other than PRNP that might be responsible of this variability. Methods We searched for other genes by performing genome-wide analyses (GWA) on 19 patients with genetic CJD and 18 healthy subjects carrying the E200K mutation of PRNP and belonging to the Calabrian cluster in Italy. We then validate this result in 32 patients with E200K CJD from non-cluster areas and 259 patients with sporadic CJD referred to the Italian CJD national registry. Results and conclusions We identified two single nucleotide polymorphisms on the CYP4X1 gene locus as candidate disease modifiers in patients with E200K CJD of the cluster area and confirmed this finding in 32 patients with E200K CJD from non-cluster areas and 259 patients with sporadic CJD. Our results indicate that the CYP4X1 gene modulates the onset of disease in patients with E200K genetic and sporadic CJD. This finding improves our understanding on the pathogenesis of CJD, suggests new targets for developing novel therapeutic strategies and might be useful for the stratification of patients in future preventive treatment trials.

Two novel PRNP truncating mutations broaden the spectrum of prion amyloidosis

Truncating mutations in PRNP have been associated with heterogeneous phenotypes ranging from chronic diarrhea and neuropathy to dementia, either rapidly or slowly progressive. We identified novel PRNP stop‐codon mutations (p.Y163X, p.Y169X) in two Italian kindreds. Disease typically presented in the third or fourth decade with progressive autonomic failure and diarrhea. Moreover, one proband (p.Y163X) developed late cognitive decline, whereas some of his relatives presented with isolated cognitive and psychiatric symptoms. Our results strengthen the link between PRNP truncating mutations and systemic abnormal PrP deposition and support a wider application of PRNP screening to include unsolved cases of familial autonomic neuropathy.