Transcribed-ultraconserved regions (T-UCRs) are long non-coding RNAs (lncRNA) encoded by a subset of long ultraconserved stretches in the human genome. Recent studies revealed that the expression of several T-UCRs is altered in cancer and growing evidences underline the importance of T-UCRs in oncogenesis, offering also potential new strategies for diagnosis and prognosis. We found that overexpression of one specific T-UCRs named uc.63 is associated with bad outcome in luminal A subtype of breast cancer patients. uc.63 is localized in the third intron of exportin-1 gene (XPO1) and is transcribed in the same orientation of its host gene. Interestingly, silencing of uc.63 induces apoptosis in vitro. However, silencing of host gene XPO1 does not cause the same effect suggesting that the transcription of uc.63 is independent of XPO1. Our results reveal an important role of uc.63 in promoting breast cancer cells survival and offer the prospect to identify a signature associated with poor prognosis.

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.

Loss-of-function mutations in APOPT1, a gene exclusively found in higher eukaryotes, cause a characteristic type of cavitating leukoencephalopathy associated with mitochondrial cytochrome c oxidase (COX) deficiency. Although the genetic association of APOPT1 pathogenic variants with isolated COX defects is now clear, the biochemical link between APOPT1 function and COX has remained elusive. We investigated the molecular role of APOPT1 using different approaches. First, we generated an Apopt1 knockout mouse model which shows impaired motor skills, e.g., decreased motor coordination and endurance, associated with reduced COX activity and levels in multiple tissues. In addition, by achieving stable expression of wild-type APOPT1 in control and patient-derived cultured cells we ruled out a role of this protein in apoptosis and established instead that this protein is necessary for proper COX assembly and function. On the other hand, APOPT1 steady-state levels were shown to be controlled by the ubiquitination-proteasome system (UPS). Conversely, in conditions of increased oxidative stress, APOPT1 is stabilized, increasing its mature intramitochondrial form and thereby protecting COX from oxidatively induced degradation.

Accumulating evidence has proved that deregulation of ΔNp63 expression plays an oncogenic role in head and neck squamous cell carcinomas (HNSCCs). Besides p63, the type 1-insulin-like growth factor (IGF) signalling pathway has been implicated in HNSCC development and progression. Most insulin/IGF1 signalling converges intracellularly onto the protein adaptor insulin receptor substrate-1 (IRS-1) that transmits signals from the receptor to downstream effectors, including the PI3K/AKT and the MAPK kinase pathways, which, ultimately, promote proliferation, invasion, and cell survival. Here we report that p63 directly controls IRS1 transcription and cellular abundance and fosters the PI3K/AKT and MAPK downstream signalling pathways. Inactivation of ΔNp63 expression indeed reduces tumour cell responsiveness to IGF1 stimulation, and inhibits the growth potential of HNSCC cells. In addition, a positive correlation was observed between p63 and IRS1 expression in human HNSCC tissue arrays and in publicly available gene expression data. Our findings indicate that aberrant expression of ΔNp63 in HNSSC may act as an oncogenic stimulus by altering the IGF signalling pathway.

Exon skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne muscular dystrophy (DMD): the rational being to convert severe Duchenne forms into milder Becker ones. Here, we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Δ44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover timing of myogenic marker expression, to relocalize neuronal nitric oxide synthase (nNOS) and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway. Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild-type functions. Among them, efficient assembly of the dystrophin-associated protein complex (DAPC) and nNOS localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to relocalize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway.

Mutations in the TMEM70 are the most common cause of nuclear ATP synthase deficiency resulting in a distinctive phenotype characterized by severe neonatal hypotonia, hypertrophic cardiomyopathy (HCMP), facial dysmorphism, severe lactic acidosis, hyperammonemia and 3-methylglutaconic aciduria (3-MGA).

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with impact on neuronal function and survival in the developing brain.

The neuron-restricted isoform 3 of the plasma membrane Ca2+ ATPase plays a major role in the regulation of Ca2+ homeostasis in the brain, where the precise control of Ca2+ signaling is a necessity. Several function-affecting genetic mutations in the PMCA3 pump associated to X-linked congenital cerebellar ataxias have indeed been described. Interestingly, the presence of co-occurring mutations in additional genes suggest their synergistic action in generating the neurological phenotype as digenic modulators of the role of PMCA3 in the pathologies. Here we report a novel PMCA3 mutation (G733R substitution) in the catalytic P-domain of the pump in a patient affected by non-progressive ataxia, muscular hypotonia, dysmetria and nystagmus. Biochemical studies of the pump have revealed impaired ability to control cellular Ca2+ handling both under basal and under stimulated conditions. A combined analysis by homology modeling and molecular dynamics have revealed a role for the mutated residue in maintaining the correct 3D configuration of the local structure of the pump. Mutation analysis in the patient has revealed two additional function-impairing compound heterozygous missense mutations (R123Q and G214S substitution) in phosphomannomutase 2 (PMM2), a protein that catalyzes the isomerization of mannose 6-phosphate to mannose 1-phosphate. These mutations are known to be associated with Type Ia congenital disorder of glycosylation (PMM2-CDG), the most common group of disorders of N-glycosylation. The findings highlight the association of PMCA3 mutations to cerebellar ataxia and strengthen the possibility that PMCAs act as digenic modulators in Ca2+-linked pathologies.

ITPR1 encodes an intracellular receptor for inositol 1,4,5-trisphosphate (InsP3) which is highly expressed in the cerebellum and is involved in the regulation of Ca2+ homeostasis. Missense mutations in the InsP3-binding domain (IRBIT) of ITPR1 are frequently associated with early onset cerebellar atrophy. Gillespie syndrome is characterized by congenital ataxia, mild to moderate intellectual disability and iris hypoplasia. Dominant or recessive ITPR1 mutations have been recently associated with this form of syndromic ataxia. We performed next generation sequencing in two simplex families with Gillespie syndrome and identified de novo pathological mutations localized in the C-terminal channel domain of ITPR1 in both patients: a recurrent deletion (p.Lys2596del) and a novel missense mutation (p.Asn2576Ile) close to a point of constriction in the Ca2+ pore. Our study expands the mutational spectrum of ITPR1 and confirms that ITPR1 screening should be implemented in patients with congenital cerebellar ataxia with or without iris hypoplasia.

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder of X-linked inheritance caused by a mutation in the ABCD1 gene which determines an accumulation of long-chain fatty acids in plasma and tissues. Recent evidence shows that oxidative stress may be a hallmark in the pathogenesis of X-ALD and glutathione plays an important role in the defense against free radicals. In this study we have analyzed glutathione homeostasis in lymphocytes of 14 patients with X-ALD and evaluated the balance between oxidized and reduced forms of glutathione, in order to define the role of this crucial redox marker in this condition.

Barth syndrome (BS) is an X-linked infantile-onset cardioskeletal disease characterized by cardiomyopathy, hypotonia, growth delay, neutropenia and 3-methylglutaconic aciduria. It is caused by mutations in the TAZ gene encoding tafazzin, a protein involved in the metabolism of cardiolipin, a mitochondrial-specific phospholipid involved in mitochondrial energy production.

Vici syndrome is a recessively inherited multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. To investigate the molecular basis of Vici syndrome, we carried out exome and Sanger sequence analysis in a cohort of 18 affected individuals. We identified recessive mutations in EPG5 (previously KIAA1632), indicating a causative role in Vici syndrome. EPG5 is the human homolog of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes. Further studies showed a severe block in autophagosomal clearance in muscle and fibroblasts from individuals with mutant EPG5, resulting in the accumulation of autophagic cargo in autophagosomes. These findings position Vici syndrome as a paradigm of human multisystem disorders associated with defective autophagy and suggest a fundamental role of the autophagy pathway in the immune system and the anatomical and functional formation of organs such as the brain and heart.

The formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation. Here, we report the reciprocal effect on keratin expression of ΔNp63, pivotal in normal epidermal morphogenesis and maintenance, and Skn-1a/Oct-11, a POU transcription factor that triggers and regulates the differentiation of keratinocytes. The expression of Skn-1a markedly downregulated ΔNp63-driven K14 expression in luciferase reporter assays. The extent of downregulation was comparable to the inhibition of Skn-1a-mediated K10 expression upon expression of ΔNp63. ΔNp63, mutated in the protein-protein interaction domain (SAM domain; mutated in human ectodermal dysplasia syndrome), was significantly less effecting in downregulating K10, raising the possibility of a direct interaction among Skn-1a and ΔNp63. Immunolocalization in human skin biopsies revealed that the expression of the two transcription factors is partially overlapping. Co-immunoprecipitation experiments did not, however, demonstrate a direct interaction between ΔNp63 and Skn-1a, suggesting that the antagonistic effects of Skn-1a and p63 on keratin promoter transactivation is probably through competition for overlapping binding sites on target gene promoter or through an indirect interaction.

NG2, the rat homologue of the human melanoma chondroitin sulfate proteoglycan (MCSP), is a ligand for collagen VI (COL6). We have examined skeletal muscles of patients affected by Ullrich scleroatonic muscular dystrophy (UCMD), an inherited syndrome caused by COL6 genes mutations. A significant decrease of NG2 immunolabeling was found in UCMD myofibers, as well as in skeletal muscle and cornea of COL6 null-mice. In UCMD muscles, truncated NG2 core protein isoforms were detected. However, real-time RT-PCR analysis revealed marked increase in NG2 mRNA content in UCMD muscle compared to controls. We hypothesize that NG2 immunohistochemical and biochemical behavior may be compromised owing to the absence of its physiological ligand. MCSP/NG2 proteoglycan may be considered an important receptor mediating COL6-sarcolemma interactions, a relationship that is disrupted by the pathogenesis of UCMD muscle.

NG2 is the rat homologue of the human melanoma chondroitin sulfate proteoglycan (MCSP) preferentially expressed in dividing progenitor cells of the glial and mesenchymal lineage but downregulated after differentiation. It has recently been demonstrated that MCSP/NG2 expression is not restricted to mitotic or malignant cells. We show that MCSP/NG2 expression is detectable in the sarcolemma, and in the neuromuscular junction of human postnatal skeletal muscle, and it gradually reduces with advancing age. In human and murine myogenic cell lines, we found no clear differences in MCSP/NG2 expression between myoblasts and myotubes. Reduced levels of the core protein were found in merosin-negative congenital muscular dystrophy (MDC1A). Duchenne muscular dystrophy patients muscles exhibited an overexpression of the MCSP/NG2 core protein. In gamma-sarcoglycanopathy and calpainopathy, MCSP/NG2 upregulation was restricted to regenerating myofibers. We demonstrate that MCSP/NG2 is expressed in differentiated myofibers, and appears to have a role in the pathogenesis of MDC1A and severe dystrophinopathies.

Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions.

The mitochondrial Elongation Factor Tu (EF-Tu), encoded by the TUFM gene, is a highly conserved GTPase, which is part of the mitochondrial protein translation machinery. In its activated form it delivers the aminoacyl-tRNAs to the A site of the mitochondrial ribosome. We report here on a baby girl with severe infantile macrocystic leukodystrophy with micropolygyria and a combined defect of complexes I and IV in muscle biopsy, caused by a novel mutation identified in TUFM. Using human mutant cells and the yeast model, we demonstrate the pathological role of the novel variant. Moreover, results of a molecular modeling study suggest that the mutant is inactive in mitochondrial polypeptide chain elongation, probably as a consequence of its reduced ability to bind mitochondrial aa-tRNAs. Four patients have so far been described with mutations in TUFM, and, following the first description of the disease in a single patient, we describe similar clinical and neuroradiological features in an additional patient.

Scoliosis is the most debilitating issue in SMA type 2 patients. No evidence confirms the efficacy of Garches braces (GB) to delay definitive spinal fusion.

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown-Vialetto-Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell-selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy-lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.