Functional analysis of a conserved site mutation in the DNA end processing enzyme PNKP leading to ataxia with oculomotor apraxia type 4 in humans.

Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells-a potential cause of neurodegeneration in AOA4 patients.

An IL-5 Single-Nucleotide Polymorphism Influences Neuroinflammation and Prospective Disease Activity in Multiple Sclerosis.

(1) Multiple sclerosis (MS) is identified by a complex interaction between central inflammation and neurodegeneration. Genetic individual variability could play a significative role in clinical presentation. The interleukin-5 (IL-5) rs2069812 single-nucleotide polymorphism (SNP) seems to define the clinical course of Th2 autoimmune diseases, while its role in MS has never been investigated. (2) In a group of 230 patients diagnosed with relapsing-remitting MS (RR-MS) or progressive MS (P-MS) and controls (IC), rs2069812 polymorphism, cerebrospinal fluid (CSF) levels of inflammatory mediators, and clinical and demographic characteristics were determined. In RR-MS patients, No Evidence of Disease Activity (NEDA-3) at three years of follow-up was detected. (3) We identified higher levels of proinflammatory cytokines, particularly IL-2 (median [IQR], RR-MS = 0.2 [0-0.7]; P-MS = 0.1 [0-1.6]; IC = 0.1 [0.0-0.1]; p < 0.005), IL-6 (RR-MS = 0.9 [0.3-2.3]; P-MS = 0.8 [0.1-2.7]; IC = 0.1 [0.0-0.5]; p < 0.005), IL-12 (RR-MS = 0.5 [0-1.1]; P-MS = 0.5 [0-1.1]; IC = 0.0 [0.0-0.3]; p < 0.005), and GM-CSF (RR-MS = 15.6 [4.8-26.4]; P-MS = 14 [3.3-29.7]; IC = 8.9 [4.7-11.7]; p < 0.005) in MS patients compared with IC. Conversely, anti-inflammatory cytokines, specifically IL-5 (RR-MS = 0.65 [0-2.4]; P-MS = 0.1 [0-0.8]; IC = 1.7 [0.6-2.8]; p < 0.005) and IL-1ra (RR-MS = 14.7 [4.9-26.4]; P-MS = 13.1 [4.7-22.2]; IC = 27.8 [17.7-37.6]; p < 0.005) were higher in controls. According to rs2069812, in MS patients, the T-allele was associated with higher concentrations of proinflammatory mediators (IL-2, CT/TT = 0.2 [0.0-2.0]; CC = 0.1 [0.0-0.4], p = 0.015; IL-6, CT/TT = 1.2 [0.4-3.2] vs. CC = 0.7 [0.1-1.7], p = 0.007; IL-15, CT/TT = 0.1 [0.0-9.5] vs. CC = 0.0 [0.0-0.1], p = 0.019; and GM-CSF, CT/TT = 0.1 [0.0-0.6] vs. CC = 0.05 [0.0-0.1], p < 0.001), and CC was associated with anti-inflammatory mediators (IL-5, CT/TT = 0.03 [0.0-1.9] vs. CC = 1.28 [0.0-2.7], p = 0.001; IL-1ra, CT/TT = 12.1 [4.1-25.9] vs. CC = 18.1 [12.1-26.9], p = 0.006). We found the same differences in RR-MS patients (IL-2, T-allele median [IQR] = 0.3 [0.0-2.0] vs. C-allele, median [IQR] = 0.04 [0.0-0.3]; p = 0.005; IL-6, T-allele, median [IQR] = 1.3 [0.4-3.3] vs. C-allele, median [IQR] = 0.6 [0.03-1.5]; p = 0.001; IL-15, T-allele, median [IQR] = 0.1 [0.0-9.5] vs. C-allele, median [IQR] = 0.0 [0.0-0.1]; p = 0.008; GM-CSF, T-allele, median [IQR] = 0.1 [0.0-97.9] vs. C-allele, median [IQR] = 0.0 [0.0-0.001]; p < 0.001; IL-5, T-allele, median [IQR] = 0.02 [0.0-2.2] vs. C-allele, median [IQR] = 1.5 [0.0-2.9]; p = 0.016; and IL-1ra, T-allele, median [IQR] = 12.1 [4.3-26.4] vs. C-allele, median [IQR] = 18.5 [12.7-28.3]; p = 0.006) but not in P-MS, except for IL-5 (T-allele, median [IQR] = 0.1 [0-0.23] vs. C-allele, median [IQR] = 0.6 [0.0-2.5]; p = 0.022). Finally, we identified an association between CC in RR-MS patients and NEDA-3 after three years of follow-up (p = 0.007). (4) We describe, for the first time, the role of an SNP of the IL-5 gene in regulating central neuroinflammation and influencing clinical course in MS patients.

Interaction between miR-142-3p and BDNF Val/Met Polymorphism Regulates Multiple Sclerosis Severity.

MiR-142-3p has recently emerged as key factor in tailoring personalized treatments for multiple sclerosis (MS), a chronic autoimmune demyelinating disease of the central nervous system (CNS) with heterogeneous pathophysiology and an unpredictable course. With its involvement in a detrimental regulatory axis with interleukin-1beta (IL1ß), miR-142-3p orchestrates excitotoxic synaptic alterations that significantly impact both MS progression and therapeutic outcomes. In this study, we investigated for the first time the influence of individual genetic variability on the miR-142-3p excitotoxic effect in MS. We specifically focused on the single-nucleotide polymorphism Val66Met (rs6265) of the brain-derived neurotrophic factor (BDNF) gene, known for its crucial role in CNS functioning. We assessed the levels of miR-142-3p and IL1ß in cerebrospinal fluid (CSF) obtained from a cohort of 114 patients with MS upon diagnosis. By stratifying patients according to their genetic background, statistical correlations with clinical parameters were performed. Notably, in Met-carrier patients, we observed a decoupling of miR-142-3p levels from IL1ß levels in the CSF, as well as from of disease severity (Expanded Disability Status Score, EDSS; Multiple Sclerosis Severity Score, MSSS; Age-Related Multiple Sclerosis Severity Score, ARMSS) and progression (Progression Index, PI). Our discovery of the interference between BDNF Val66Met polymorphism and the synaptotoxic IL1ß-miR-142-3p axis, therefore hampering miR-142-3p action on MS course, provides valuable insights for further development of personalized medicine in the field.

Harmonizing Genetic Testing for Parkinson's Disease: Results of the PARKNET Multicentric Study.

BACKGROUND AND OBJECTIVE: Early-onset Parkinson's disease (EOPD) commonly recognizes a genetic basis; thus, patients with EOPD are often addressed to diagnostic testing based on next-generation sequencing (NGS) of PD-associated multigene panels. However, NGS interpretation can be challenging in a diagnostic setting, and few studies have addressed this issue so far. METHODS: We retrospectively collected data from 648 patients with PD with age at onset younger than 55 years who underwent NGS of a minimal shared panel of 15 PD-related genes, as well as PD-multiplex ligation-dependent probe amplification in eight Italian diagnostic laboratories. Data included a minimal clinical dataset, the complete list of variants included in the diagnostic report, and final interpretation (positive/negative/inconclusive). Patients were further stratified based on age at onset ≤40 years (very EOPD, n = 157). All variants were reclassified according to the latest American College of Medical Genetics and Genomics criteria. For classification purposes, PD-associated GBA1 variants were considered diagnostic. RESULTS: In 186 of 648 (29%) patients, the diagnostic report listed at least one variant, and the outcome was considered diagnostic (positive) in 105 (16%). After reanalysis, diagnosis changed in 18 of 186 (10%) patients, with 5 shifting from inconclusive to positive and 13 former positive being reclassified as inconclusive. A definite diagnosis was eventually reached in 97 (15%) patients, of whom the majority carried GBA1 variants or, less frequently, biallelic PRKN variants. In 89 (14%) cases, the genetic report was inconclusive. CONCLUSIONS: This study attempts to harmonize reporting of PD genetic testing across several diagnostic labs and highlights current difficulties in interpreting genetic variants emerging from NGS-multigene panels, with relevant implications for counseling. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genetic regulation of IL-8 influences disease presentation of multiple sclerosis.

BACKGROUND: Individual genetic variability may influence the course of multiple sclerosis (MS). The interleukin (IL)-8C>T rs2227306 single nucleotide polymorphism (SNP) regulates IL-8 activity in other clinical conditions; however, its role in MS has never been investigated. OBJECTIVES: To explore the association between IL-8 SNP rs2227306, cerebrospinal fluid (CSF) IL-8 concentrations, clinical, and radiological characteristics in a group of newly diagnosed MS patients. METHODS: In 141 relapsing-remitting (RR)-MS patients, rs2227306 polymorphism, CSF levels of IL-8, clinical, and demographical characteristics were determined. In 50 patients, structural magnetic resonance imaging (MRI) measures were also assessed. RESULTS: An association between CSF IL-8 and Expanded Disability Status Scale (EDSS) at diagnosis was found in our set of patients (r = 0.207, p = 0.014). CSF IL-8 concentrations were significantly higher in patients carrying the T variant of rs2227306 (p = 0.004). In the same group, a positive correlation emerged between IL-8 and EDSS (r = 0.273, p = 0.019). Finally, a negative correlation between CSF levels of IL-8 and cortical thickness emerged in rs2227306T carriers (r = -0.498, p = 0.005). CONCLUSION: We describe for the first time a role of SNP rs2227306 of IL-8 gene in regulating the expression and the activity of this inflammatory cytokine in MS.

Decipher non-canonical SPAST splicing mutations with the help of functional assays in patients affected by spastic paraplegia 4 (SPG4).

Flowchart showing the molecular approach used to decipher the non-canonical splicing mutations.

Improving clinical interpretation of five KRIT1 and PDCD10 intronic variants.

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system which may occur sporadically or segregate within families due to heterozygous variants in KRIT1/CCM1, MGC4607/CCM2 or PDCD10/CCM3. Intronic variants are not uncommon in familial CCM, but their clinical interpretation is often hampered by insufficient data supporting in silico predictions. Here, the mRNA analysis for two intronic unpublished variants (KRIT1 c.1147-7 T > G and PDCD10 c.395 + 2 T > G) and three previously published variants in KRIT1 but without data supporting their effects was carried out. This study demonstrated that all variants can induce a frameshift with the lack of residues located in the C-terminal regions and involved in protein-protein complex formation, which is essential for vascular homeostasis. These results support the introduction of mRNA analysis in the diagnostic pathway of familial CCM and expand the knowledge of abnormal splicing patterning in this disorder.

An attempt to dissect a peripheral marker based on cell pathology in Parkinson's disease.

Peripheral markers in Parkinson's disease (PD) represent a hot issue to provide early diagnosis and assess disease progression. The gold standard marker of PD should feature the same reliability as the pathogenic alteration, which produces the disease itself. PD is foremost a movement disorder produced by a loss of nigrostriatal dopamine innervation, in which striatal dopamine terminals are always markedly reduced in PD patients to an extent, which never overlaps with controls. Similarly, a reliable marker of PD should possess such a non-overlapping feature when compared with controls. In the present study, we provide a novel pathological hallmark, the autophagosome, which in each PD patient was always suppressed compared with each control subject. Autophagosomes were counted as microtubule-associated proteins 1A/1B light chain 3B (LC3)-positive vacuoles at ultrastructural morphometry within peripheral (blood) blood mononuclear cells (PBMC). This also provides the gold standard to assess the autophagy status. Since autophagy may play a role in the pathogenesis of PD, autophagosomes may be a disease marker, while participating in the biology of the disease. Stoichiometric measurement of α-synuclein despite significantly increased in PD patients, overlapped between PD and control patients. Although the study need to be validated in large populations, the number of autophagy vacuoles is neither related with therapy (the amount was similarly suppressed in a few de novo patients), nor the age in PD or controls.

Rapamycin Ameliorates Defects in Mitochondrial Fission and Mitophagy in Glioblastoma Cells.

Glioblastoma (GBM) cells feature mitochondrial alterations, which are documented and quantified in the present study, by using ultrastructural morphometry. Mitochondrial impairment, which roughly occurs in half of the organelles, is shown to be related to mTOR overexpression and autophagy suppression. The novelty of the present study consists of detailing an mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission. These phenomena contribute to explain the increase in altered mitochondria reported here. Administration of the mTOR inhibitor rapamycin rescues mitochondrial alterations. In detail, rapamycin induces the expression of genes promoting mitophagy (PINK1, PARKIN, ULK1, AMBRA1) and mitochondrial fission (FIS1, DRP1). This occurs along with over-expression of VPS34, an early gene placed upstream in the autophagy pathway. The topographic stoichiometry of proteins coded by these genes within mitochondria indicates that, a remarkable polarization of proteins involved in fission and mitophagy within mitochondria including LC3 takes place. Co-localization of these proteins within mitochondria, persists for weeks following rapamycin, which produces long-lasting mitochondrial plasticity. Thus, rapamycin restores mitochondrial status in GBM cells. These findings add novel evidence about mitochondria and GBM, while fostering a novel therapeutic approach to restore healthy mitochondria through mTOR inhibition.

A novel POLR3A genotype leads to leukodystrophy type-7 in two siblings with unusually late age of onset.

BACKGROUND: Leukodystrophies are familial heterogeneous disorders primarily affecting the white matter, which are defined as hypomyelinating or demyelinating based on disease severity as assessed at MRI. Recently, a group of clinically overlapping hypomyelinating leukodystrophies (HL) has been associated with mutations in RNA polymerase III enzymes (Pol III) subunits. CASE PRESENTATION: In this manuscript, we describe two Italian siblings carrying a novel POLR3A genotype. MRI imaging, genetic analysis, and clinical data led to diagnosing HL type 7. The female sibling, at the age of 34, is tetra-paretic and suffers from severe cognitive regression. She had a disease onset at the age of 19, characterized by slow and progressive cognitive impairment associated with gait disturbances and amenorrhea. The male sibling was diagnosed during an MRI carried out for cephalalgia at the age of 41. After 5 years, he developed mild cognitive impairment, dystonia with 4-limb hypotonia, and moderate dysmetria with balance and gait impairment. CONCLUSIONS: The present study provides the first evidence of unusually late age of onset in HL, describing two siblings with a novel POLR3A genotype which showed the first symptoms at the age of 41 and 19, respectively. This provides a powerful insight into clinical heterogeneity and genotype-phenotype correlation in POLR3A related HL.

Promiscuous Roles of Autophagy and Proteasome in Neurodegenerative Proteinopathies.

Alterations in autophagy and the ubiquitin proteasome system (UPS) are commonly implicated in protein aggregation and toxicity which manifest in a number of neurological disorders. In fact, both UPS and autophagy alterations are bound to the aggregation, spreading and toxicity of the so-called prionoid proteins, including alpha synuclein (α-syn), amyloid-beta (Aß), tau, huntingtin, superoxide dismutase-1 (SOD-1), TAR-DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS). Recent biochemical and morphological studies add to this scenario, focusing on the coordinated, either synergistic or compensatory, interplay that occurs between autophagy and the UPS. In fact, a number of biochemical pathways such as mammalian target of rapamycin (mTOR), transcription factor EB (TFEB), Bcl2-associated athanogene 1/3 (BAG3/1) and glycogen synthase kinase beta (GSk3ß), which are widely explored as potential targets in neurodegenerative proteinopathies, operate at the crossroad between autophagy and UPS. These biochemical steps are key in orchestrating the specificity and magnitude of the two degradation systems for effective protein homeostasis, while intermingling with intracellular secretory/trafficking and inflammatory pathways. The findings discussed in the present manuscript are supposed to add novel viewpoints which may further enrich our insight on the complex interactions occurring between cell-clearing systems, protein misfolding and propagation. Discovering novel mechanisms enabling a cross-talk between the UPS and autophagy is expected to provide novel potential molecular targets in proteinopathies.

Dissecting Molecular Features of Gliomas: Genetic Loci and Validated Biomarkers.

Recently, several studies focused on the genetics of gliomas. This allowed identifying several germline loci that contribute to individual risk for tumor development, as well as various somatic mutations that are key for disease classification. Unfortunately, none of the germline loci clearly confers increased risk per se. Contrariwise, somatic mutations identified within the glioma tissue define tumor genotype, thus representing valid diagnostic and prognostic markers. Thus, genetic features can be used in glioma classification and guided therapy. Such copious genomic variabilities are screened routinely in glioma diagnosis. In detail, Sanger sequencing or pyrosequencing, fluorescence in-situ hybridization, and microsatellite analyses were added to immunohistochemistry as diagnostic markers. Recently, Next Generation Sequencing was set-up as an all-in-one diagnostic tool aimed at detecting both DNA copy number variations and mutations in gliomas. This approach is widely used also to detect circulating tumor DNA within cerebrospinal fluid from patients affected by primary brain tumors. Such an approach is providing an alternative cost-effective strategy to genotype all gliomas, which allows avoiding surgical tissue collection and repeated tumor biopsies. This review summarizes available molecular features that represent solid tools for the genetic diagnosis of gliomas at present or in the next future.

Quantitative Ultrastructural Morphometry and Gene Expression of mTOR-Related Mitochondriogenesis within Glioblastoma Cells.

In glioblastoma (GBM) cells, an impairment of mitochondrial activity along with autophagy suppression occurs. Autophagy suppression in GBM promotes stemness, invasion, and poor prognosis. The autophagy deficit seems to be due, at least in part, to an abnormal up-regulation of the mammalian target of rapamycin (mTOR), which may be counteracted by pharmacological mTORC1 inhibition. Since autophagy activation is tightly bound to increased mitochondriogenesis, a defect in the synthesis of novel mitochondria is expected to occur in GBM cells. In an effort to measure a baseline deficit in mitochondria and promote mitochondriogenesis, the present study used two different GBM cell lines, both featuring mTOR hyperactivity. mTORC1 inhibition increases the expression of genes and proteins related to autophagy, mitophagy, and mitochondriogenesis. Autophagy activation was counted by RT-PCR of autophagy genes, LC3- immune-fluorescent puncta and immune-gold, as well as specific mitophagy-dependent BNIP3 stoichiometric increase in situ, within mitochondria. The activation of autophagy-related molecules and organelles after rapamycin exposure occurs concomitantly with progression of autophagosomes towards lysosomes. Remarkably, mitochondrial biogenesis and plasticity (increased mitochondrial number, integrity, and density as well as decreased mitochondrial area) was long- lasting for weeks following rapamycin withdrawal.

Facioscapulohumeral muscular dystrophy (FSHD) molecular diagnosis: from traditional technology to the NGS era.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic neuromuscular disorder which mainly affects the muscles of the face, shoulder, and upper arms. FSHD is generally associated with the contraction of D4Z4 macrosatellite repeats on 4q35 chromosome or mutations in SMCHD1, which are responsible of the toxic expression of DUX4 in muscle tissue. Despite the recent application of NGS techniques in the clinical practice, the molecular diagnosis of FSHD is still performed with dated techniques such as Southern blotting. The diagnosis of FSHD requires therefore specific skills on both modern and less modern analytical protocols. Considering that clinical and molecular diagnosis of FSHD is challenging, it is not surprising that only few laboratories offer a comprehensive characterization of FSHD, which requires the education of professionals on traditional techniques even in the era of NGS. In conclusion, the study of FSHD provides an excellent example of using classical and modern molecular technologies which are equally necessary for the analysis of DNA repetitive traits associated with specific disorders.

Migrainous Infarction in a Patient With Sporadic Hemiplegic Migraine and Cystic Fibrosis: A 99mTc-HMPAO Brain SPECT Study.

Genetic mutations of sporadic hemiplegic migraine (SHM) are mostly unknown. SHM pathophysiology relies on cortical spreading depression (CSD), which might be responsible for ischemic brain infarction. Cystic fibrosis (CF) is caused by a monogenic mutation of the chlorine transmembrane conductance regulator (CFTR), possibly altering brain excitability. We describe the case of a patient with CF, who had a migrainous stroke during an SHM attack. A 32-year-old Caucasian male was diagnosed with CF, with heterozygotic delta F508/unknown CFTR mutation. The patient experiences bouts of coughing sometimes triggering SHM attacks with visual phosphenes, aphasia, right-sided paresthesia, and hemiparesis. He had a 48-hour hemiparesis triggered by a bout of coughing with hemoptysis, loss of consciousness, and severe hypoxia-hypercapnia. MRI demonstrated transient diffusion hyperintensity in the left frontal-parietal-occipital regions resulting in a permanent infarction in the primary motor area. Later, a brain perfusion SPECT showed persistent diffuse hypoperfusion in the territories involved in diffusion-weighted imaging alteration. Migrainous infarction, depending on the co-occurrence of 2 strictly related phenomena, CSD and hypoxia, appears to be the most plausible explanation. Brain SPECT hypoperfusion suggests a more extensive permanent neuronal loss in territories affected by aura. CF may be then a risk factor for hemiplegic migraine and stroke since bouts of coughing can facilitate brain hypoxia, triggering auras.

Unusual Segregation of APP Mutations in Monogenic Alzheimer Disease.

APP gene mutations causing Alzheimer disease (AD) segregate in an autosomal dominant pattern. We report on a 40-year-old woman with a severe cognitive decline starting at 36 years, while her affected relatives presented symptoms onset in the 6th decade. The proband carried an APP missense variant in homozygous state (NM_000484.4: c.2032G>A; NP_000475.1: p.Asp678Asn; rs63750064) and showed a more severe clinical picture than the other AD relatives, as regards the age of onset and the rate of disease progression. This mutation behaves as a semi-dominant trait. The very rare chance of studying APP mutations in the homozygous state demonstrates they are not always dominant and other segregation models are possible.

Corticosterone Upregulates Gene and Protein Expression of Catecholamine Markers in Organotypic Brainstem Cultures.

Glucocorticoids are produced by the adrenal cortex and regulate cell metabolism in a variety of organs. This occurs either directly, by acting on specific receptors in a variety of cells, or by stimulating catecholamine expression within neighbor cells of the adrenal medulla. In this way, the whole adrenal gland may support specific metabolic requirements to cope with stressful conditions from external environment or internal organs. In addition, glucocorticoid levels may increase significantly in the presence of inappropriate secretion from adrenal cortex or may be administered at high doses to treat inflammatory disorders. In these conditions, metabolic alterations and increased blood pressure may occur, although altered sleep-waking cycle, anxiety, and mood disorders are frequent. These latter symptoms remain unexplained at the molecular level, although they overlap remarkably with disorders affecting catecholamine nuclei of the brainstem reticular formation. In fact, the present study indicates that various doses of glucocorticoids alter the expression of genes and proteins, which are specific for reticular catecholamine neurons. In detail, corticosterone administration to organotypic mouse brainstem cultures significantly increases Tyrosine hydroxylase (TH) and Dopamine transporter (DAT), while Phenylethanolamine N-methyltransferase (PNMT) is not affected. On the other hand, Dopamine Beta-Hydroxylase (DBH) increases only after very high doses of corticosterone.

Heterozygous missense mutations in NFATC1 are associated with atrioventricular septal defect.

Atrioventricular septal defect (AVSD) may occur as part of a complex disorder (e.g., Down syndrome, heterotaxy), or as isolate cardiac defect. Multiple lines of evidence support a role of calcineurin/NFAT signaling in AVSD, and mutations in CRELD1, a protein functioning as a regulator of calcineurin/NFAT signaling have been reported in a small fraction of affected subjects. In this study, 22 patients with isolated AVSD and 38 with AVSD and heterotaxy were screened for NFATC1 gene mutations. Sequence analysis identified three missense variants in three individuals, including a subject with isolated AVSD [p.(Ala367Val)], an individual with AVSD and heterotaxy [p.(Val210Met)], and a subject with AVSD, heterotaxy, and oculo-auriculo-vertebral spectrum (OAVS) [p.(Ala696Thr)], respectively. The latter was also heterozygous for a missense change in TBX1 [p.(Pro86Leu)]. Targeted resequencing of genes associated with AVSD, heterotaxy, or OAVS excluded additional hits in the three mutation-positive subjects. Functional characterization of NFATC1 mutants documented defective nuclear translocation and decreased transcriptional transactivation activity. When expressed in zebrafish, the three NFATC1 mutants caused cardiac looping defects and altered atrioventricular canal patterning, providing evidence of their functional relevance in vivo. Our findings support a role of defective NFATC1 function in the etiology of isolated and heterotaxy-related AVSD.

Ambiguous Effects of Autophagy Activation Following Hypoperfusion/Ischemia.

Autophagy primarily works to counteract nutrient deprivation that is strongly engaged during starvation and hypoxia, which happens in hypoperfusion. Nonetheless, autophagy is slightly active even in baseline conditions, when it is useful to remove aged proteins and organelles. This is critical when the mitochondria and/or proteins are damaged by toxic stimuli. In the present review, we discuss to that extent the recruitment of autophagy is beneficial in counteracting brain hypoperfusion or, vice-versa, its overactivity may per se be detrimental for cell survival. While analyzing these opposite effects, it turns out that the autophagy activity is likely not to be simply good or bad for cell survival, but its role varies depending on the timing and amount of autophagy activation. This calls for the need for an appropriate autophagy tuning to guarantee a beneficial effect on cell survival. Therefore, the present article draws a theoretical pattern of autophagy activation, which is hypothesized to define the appropriate timing and intensity, which should mirrors the duration and severity of brain hypoperfusion. The need for a fine tuning of the autophagy activation may explain why confounding outcomes occur when autophagy is studied using a rather simplistic approach.

Severe and rapidly-progressive Lafora disease associated with NHLRC1 mutation: a case report.

Lafora disease (LD), also known as progressive myoclonic epilepsy-2 (EPM2), is a rare, fatal autosomal recessive disorder typically starting during adolescence in otherwise neurologically normal individuals. It is clinically characterized by insidious of progressive neurological features including seizures, action myoclonus, visual hallucination, ataxia and dementia. Mutations in the laforin (EPM2A) gene on chromosome 6q24 or in the malin gene (NHLRC1) on chromosome 6p22 are responsible of LD phenotype. Diagnostic workup includes genetic analysis as well as axillary skin biopsy with evidence of typical periodic acid-Schiff (PAS)-positive polyglucosan inclusion bodies (Lafora bodies) in the apocrine glands and/or in the eccrine duct. Usually, genotype-phenotype correlations do not reveal substantial differences between patients carrying EPM2A and NHLRC1 mutations, but a few specific NHLRC1 mutations appear to correlate with a late onset and slow progressing LD. We report a case of LD due to compound heterozygote NHLRC1 mutation in an adolescent presenting with severe and atypical electro-clinical features, mimicking an autoimmune encephalopathy, and a rapidly progressive clinical course.