Differential allelic expression of SOS1 and hyperexpression of the activating SOS1 c.755C variant in a Noonan syndrome family

Noonan syndrome (NS) is a genetic condition characterized by congenital heart defects, short stature and characteristic facial features. We here present the case of a girl with moderate learning disabilities, delayed language development, craniofacial features and skin anomalies reminiscent of NS. After a mutation screening of the known NS genes PTPN11, SOS1, RAF1, KRAS, GRB2, BRAF and SHOC2 we found the heterozygous c.755T>C variant in SOS1 causing the p.I252T amino-acid substitution, which was considered possibly pathogenetic by bioinformatic predictions. The same variant was present in the proband's mother, displaying some NS features, and mateRNAl grandfather showing no NS traits, but also by a healthy subject in 1000 genomes project database without phenotype informations. The functional analysis revealed that SOS1 c.755C activated the RAS-ERK intracellular pathway, whereas no effects on RAC-JNK cascade have been detected. After a comparison between the sequence of SOS1 cDNA from peripheral blood and SOS1 genomic DNA, we showed for the first time a differential allelic expression of the SOS1 gene in healthy individuals, thus occurring as a physiologic condition. Interestingly, we found that the mutated allele C was 50% more expressed than the wild-type allele T in all familial carriers. The comparable amount of SOS1 mRNA between mutated individuals and the controls indicates that the variant does not affect SOS1 expression. The present study provides a first evidence of allelic imbalance of SOS1 and pinpoints this condition as a possible mechanism underlying a different penetrance of some SOS1-mutated alleles in unrelated carriers

ADAP2 in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome

Background Cardiovascular malformations have a higher incidence in patients with NF1 microdeletion syndrome compared to NF1 patients with intragenic mutation, presumably owing to haploinsufficiency of one or more genes included in the deletion interval and involved in heart development. In order to identify which genes could be responsible for cardiovascular malformations in the deleted patients, we carried out expression studies in mouse embryos and functional studies in zebrafish. Methods and results The expression analysis of three candidate genes included in the NF1 deletion interval, ADAP2, SUZ12 and UTP6, performed by in situ hybridisation, showed the expression of ADAP2 murine ortholog in heart during fundamental phases of cardiac morphogenesis. In order to investigate the role of ADAP2 in cardiac development, we performed loss-of-function experiments of zebrafish ADAP2 ortholog, adap2, by injecting two different morpholino oligos (adap2-MO and UTR-adap2-MO). adap2-MOs-injected embryos (morphants) displayed in vivo circulatory and heart shape defects. The molecular characterisation of morphants with cardiac specific markers showed that the injection of adap2-MOs causes defects in heart jogging and looping. Additionally, morphological and molecular analysis of adap2 morphants demonstrated that the loss of adap2 function leads to defective valvulogenesis, suggesting a correlation between ADAP2 haploinsufficiency and the occurrence of valve defects in NF1-microdeleted patients. Conclusions Overall, our findings indicate that ADAP2 has a role in heart development, and might be a reliable candidate gene for the occurrence of cardiovascular malformations in patients with NF1 microdeletion and, more generally, for the occurrence of a subset of congenital heart defects

Mutations and novel polymorphisms in coding regions and UTRs of CDK5R1 and OMG genes in patients with nonsyndromic mental retardation

Mental retardation (MR) is displayed by 57% of NF1 patients with microdeletion syndrome as a result of 17q11.2 region haploinsufficiency. We considered the cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) and oligodendrocyte-myelin glycoprotein (OMG) genes, mapping in the NF1 microdeleted region, as candidate genes for MR susceptibility. CDK5R1 encodes for a neurone-specific activator of cyclin-dependent kinase 5 (CDK5) involved in neuronal migration during central nervous system development. OMG encodes for an inhibitor of neurite outgrowth by the binding to the Nogo-66 receptor (RTN4R). CDK5R1 and OMG genes are characterized by large 3\u2032 and 5\u2032 untranslated regions (UTRs), where we predict the presence of several transcription/ translation regulatory elements. We screened 100 unrelated Italian patients affected by unspecific MR for mutations in CDK5R1 and OMG coding regions and in their 3\u2032 or 5\u2032 UTRs. Four novel mutations and two novel polymorphisms for CDK5R1 and three novel mutations for OMG were detected, including two missense changes (c.323C>T; A108V in CDK5R1 and c.1222A>G; T408A in OMG), one synonymous codon variant (c.532C>T; L178L in CDK5R1), four variants in CDK5R1 3\u2032UTR and two changes in OMG 5\u2032UTR. All the mutations were absent in 370 chromosomes from normal subjects. The allelic frequencies of the two novel polymorphisms in CDK5R1 3\u2032UTR were established in both 185 normal and 100 mentally retarded subjects. Prediction of mRNA and protein secondary structures revealed that two changes lead to putative structural alterations in the mutated c.2254C>G CDK5R1 3\u2032UTR and in OMG T408A gene product

ATRX mutation in two adult brothers with non-specific moderate intellectual disability identified by exome sequencing

In this report, we describe two adult brothers affected by moderate non-specific intellectual disability (ID). They showed minor facial anomalies, not clearly ascribable to any specific syndromic patterns, microcephaly, brachydactyly and broad toes. Both brothers presented seizures. Karyotype, subtelomeric and FMR1 analysis were normal in both cases. We performed array-CGH analysis that revealed no copy-number variations potentially associated with ID. Subsequent exome sequence analysis allowed the identification of the ATRX c.109C>T (p.R37X) mutation in both the affected brothers. Sanger sequencing confirmed the presence of the mutation in the brothers and showed that the mother is a healthy carrier. Mutations in the ATRX gene cause the X-linked alpha thalassemia/mental retardation (ATR-X) syndrome (MIM #301040), a severe clinical condition usually associated with profound ID, facial dysmorphism and alpha thalassemia. However, the syndrome is clinically heterogeneous and some mutations, including the c.109C>T, are associated with a broad phenotypic spectrum, with patients displaying a less severe phenotype with only mild-moderate ID. In the case presented here, exome sequencing provided an effective strategy to achieve the molecular diagnosis of ATR-X syndrome, which otherwise would have been difficult to consider due to the mild non-specific phenotype and the absence of a family history with typical severe cases

The miR-15/107 Family of microRNA Genes Regulates CDK5R1/p35 with Implications for Alzheimer’s Disease Pathogenesis

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, the main activatory subunit of cyclin-dependent kinase 5 (CDK5). The p35/CDK5 active complex plays a fundamental role in brain development and functioning, but its deregulated activity has also been implicated in various neurodegenerative disorders, including Alzheimer\u2019s disease (AD). CDK5R1 displays a large and highly evolutionarily conserved 3\u2032-untranslated region (3\u2032-UTR), a fact that has suggested a role for this region in the post-transcriptional control of CDK5R1 expression. Our group has recently demonstrated that two miRNAs, miR-103 and miR-107, regulate CDK5R1 expression and affect the levels of p35. MiR-103 and miR-107 belong to the miR-15/107 family, a group of evolutionarily conserved miRNAs highly expressed in human cerebral cortex. In this work, we tested the hypothesis that other members of this group of miRNAs, in addition to miR-103 and miR-107, were able to modulate CDK5R1 expression. We provide evidence that several miRNAs belonging to the miR-15/107 family regulate p35 levels. BACE1 expression levels were also found to be modulated by different members of this family. Furthermore, overexpression of these miRNAs led to reduced APP phosphorylation levels at the CDK5-specific Thr668 residue. We also show that miR-15/107 miRNAs display reduced expression levels in hippocampus and temporal cortex, but not in cerebellum, of AD brains. Moreover, increased CDK5R1 mRNA levels were observed in AD hippocampus tissues. Our results suggest that the downregulation of the miR-15/107 family might have a role in the pathogenesis of AD by increasing the levels of CDK5R1/p35 and consequently enhancing CDK5 activity

MicroRNA Dysregulation in the Spinal Cord following Traumatic Injury

Spinal cord injury (SCI) triggers a multitude of pathophysiological events that are tightly regulated by the expression levels of specific genes. Recent studies suggest that changes in gene expression following neural injury can result from the dysregulation of microRNAs, short non-coding RNA molecules that repress the translation of target mRNA. To understand the mechanisms underlying gene alterations following SCI, we analyzed the microRNA expression patterns at different time points following rat spinal cord injury

Functional study of regulatory elements in CDK5R1 3’-UTR: evidence on post-transcriptional gene expression regulation

Background CDK5R1 encodes for p35, a neuron-specific activator of cyclin-dependent kinase 5 (CDK5), whose activity plays a central role in neuronal migration during central nervous system development. Cdk5r KO mice have severe cortical lamination defects and suffer from adult mortality and seizures. The active CDK5-p35 complex is involved in several processes required for central nervous system development and functioning, as axonal regeneration, cellular differentiation, neuronal apoptosis, learning and memory processes, synaptic transmission and membrane trafficking during the outgrowth of neuronal processes. Moreover, increased CDK5 activation by p25, a proteolytic fragment containing the C-terminal portion of p35, has been implicated in the pathogenesis of several neurodegenerative disorders, such as Alzheimer\u2019s disease, Parkinson\u2019s disease and amyotrophic lateral sclerosis. CDK5R1 has been proposed as a candidate gene for mental retardation susceptibility in NF1 microdeletion syndrome. CDK5R1 spans for 4.17 kb on chr 17q11.2, and its coding region (1021 bp) consists of a single exon. In addition, the CDK5R1 gene displays a large 3\u2019-untranslated region (3\u2019-UTR). The remarkable size of CDK5R1 3\u2019-UTR suggests a role in post-transcriptional regulation of CDK5R1 expression. Untranslated regions (UTRs) are known to play crucial roles in the post-transcriptional regulation of gene expression, including modulation of the transport of mRNA out of the nucleus, and of the translation efficiency, subcellular localization and stability. The importance of 3\u2019-UTRs in regulating gene expression is underlined by the finding that mutations which alter the 3\u2019-UTR can lead to serious pathology. Nucleotide patterns or motifs located in 3' UTRs can interact with specific RNA-binding proteins. The biological activity of regulatory motifs at the RNA level relies on a combination of primary and secondary structure. Interactions between sequence elements located in the 3\u2019-UTRs and specific microRNAs have also been shown to play key regulatory roles. Results The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs) and a GY-box element. The GY 12box (GTCTTCC) motif, described in many 3' 12UTRs of genes involved in Notch signalling in Drosophila is likely to be involved in the formation of RNA duplexes with complementary sequences at the 5' ends of some Drosophila microRNAs in vivo. AU-rich sequences, function as potent destabilizing elements that cause rapid decay of the respective transcript; these elements are composed of a variable number of copies of the AUUUA pentamer or UUAUUUAUU nonamer. Among the predicted AREs in CDK5R1, the nt 2659-2671 ARE shows complete identity to the consensus sequence for Class I AREs, according to the ARED 3.0 definition, is highly conserved in mammals and zebrafish and is predicted to be accessibile to the binding of trans-acting factors. The effect of the 3\u2019-UTR on gene expression was studied with the Dual-Luciferase reporter assay. The insertion of CDK5R1 3\u2019-UTR into luciferase 3\u2019-UTR caused a decreased luciferase activity and mRNA level in four transfected cell lines (SK-N-BE, SH-SY5Y, HEK-293 and MCF-7). The dissection of 3\u2019-UTR into 6 fragments (C1-6), each containing at least one predicted regulatory element, allowed us to investigate the potential role of each region. All the chimeric constructs showed, in most of the studied cell lines, a general decrease of luciferase activity. In most cases these effects are likely to involve transcript stability rather than translational repression mechanisms, since reduced reporter activity levels corresponded to reduced mRNA levels. A region (C2), leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. This fragment was dissected into smaller regions and a 65 bp (named C2.11) sequence has been identified to be responsable of the decreased gene expression of the all C2 fragment, in which none regulatory elements were predicted. The existence of a stable structural motif (forming a hairpin) was predicted by both RnaProfile and SFold programs in both the analysed entire 3'-UTR and C2.11 transcripts, and it was speculated that it may have a regulatory role. We show here that the hairpin structure within the 3'-UTR influences the expression of the luciferase reporter gene by means of generation of mutants. Lowering of luciferase levels for the construct with an intact hairpin structure in contrast with almost unchanged levels for the four mutated/deleted structures confirm the importance of this sequence and suggest that its disruption may directly affect rapid mRNA degradation. Since complementary mutations restoring the hairpin structure did not restore luciferase activity, we suggest that sequence within a structure is essential for the ability of the C2.11 fragment to reduce luciferase activity. The generation of a construct with the deletion of the canonical GY-box motif revealed the inactivity of this element in all the cell lines used for the transfection experiments. The 3\u2019 end of the transcript (C6 fragment), containing the class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. The deletion of the canonical nt 2659-2671 ARE in the C6 fragment reduced luciferase activity mRNA levels in all the analyzed cell lines, including SK-N-BE and SH-SY5Y, strongly suggesting a stabilizing role of the canonical element in neuroblastoma-derived cells through the binding of neuronal-specific stabilizing factors. We also observed the interaction of the stabilizing neuronal RNA-binding proteins nELAV with the CDK5R1 transcript in SH-SY5Y cells by immunoprecipitation and UV cross-linking experiments allowed us to observe that C1, C2 and C6 subregions show affinity for nELAV proteins in vitro. microRNA (miRNA) target site prediction with PicTar software found several target sites along the entire CDK5R1 3\u2019-UTR without clustering. Between the 20 miRNAs predicted to bind CDK5R1, miR-15a, miR-103 and miR-107 present a high number of target sites with a free energy <-20 kcal/mol which point to these three miRNAs as the most probable to be functional. Nine pre-miRNAs have been shown to be expressed in the cell lines of interest. A preliminary quantitative analysis points to an inverse correlation of expression between miR-107 and the proteic product of CDK5R1, p35, that correlates with the negative role of miRNA on expression of proteins encoded by their target transcripts. Conclusions Our findings have shown the presence of several regulatory elements in CDK5R1 3\u2019-UTR, and for a few of them we found a destabilizing or stabilizing function. The 3\u2019-UTR seems to contain some regulatory elements implicated in rapid mRNA turnover which, as a consequence, maintain the steady-state transcript at low levels, and others which have a cell-specific stabilizing effect on the transcript that may contribute to rapidly increase the expression of CDK5R1 during specific biological processes. Our findings also support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3\u2019-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3\u2019-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders. The large 3\u2019-UTR of CDK5R1 is expected to contain further cis-regulatory elements and interact with further trans-acting molecules, creating the possibility of complex gene expression modulation

Il cancro del fusto minaccia il melograno italiano

Diversi impianti di melograno stanno morendo a causa del fungo Coniella granati, patogeno per il quale è lecito attendersi una rapida diffusione