Deleterious aberrations in DNA repair genes are actionable in approximately 25% of metastatic castration-resistant prostate cancers (mCRPC) patients. Homology recombination repair (HRR) is the DNA ...damage repair (DDR) mechanism most frequently altered in prostate cancer; of note
is the most frequently altered DDR gene in this tumor. Poly ADP-ribose polymerase inhibitors showed antitumor activity with a improvement in overall survival in mCRPC carrying somatic and/or germline alterations of HHR. Germline mutations are tested on peripheral blood samples using DNA extracted from peripheral blood leukocytes, while the somatic alterations are assessed by extracting DNA from a tumor tissue sample. However, each of these genetic tests have some limitations: the somatic tests are related to the sample availability and tumor heterogeneity, while the germline testing are mainly related to the inability to detect somatic HRR mutations. Therefore, the liquid biopsy, a non-invasive and easily repeatable test compared to tissue test, could identified somatic mutation detected on the circulating tumor DNA (ctDNA) extracted from a plasma. This approach should better represent the heterogeneity of the tumor compared to the primary biopsy and maybe helpful in monitoring the onset of potential mutations involved in treatment resistance. Furthermore, ctDNA may inform about timing and potential cooperation of multiple driver genes aberration guiding the treatment options in patients with mCRPC. However, the clinical use of ctDNA test in prostate cancer compared to blood and tissue testing are currently very limited. In this review, we summarize the current therapeutic indications in prostate cancer patients with DDR deficiency, the recommendation for germline and somatic-genomic testing in advanced PC and the advantages of the use liquid biopsy in clinical routine for mCRPC.
Behavioral changes in response to stressful stimuli can be controlled via adaptive epigenetic changes in neuronal gene expression. Here we indicate a role for the transcriptional corepressor ...Lysine-Specific Demethylase 1 (LSD1) and its dominant-negative splicing isoform neuroLSD1, in the modulation of emotional behavior. In mouse hippocampus, we show that LSD1 and neuroLSD1 can interact with transcription factor serum response factor (SRF) and set the chromatin state of SRF-targeted genes early growth response 1 (egr1) and c-fos. Deletion or reduction of neuroLSD1 in mutant mice translates into decreased levels of activating histone marks at egr1 and c-fos promoters, dampening their psychosocial stress-induced transcription and resulting in low anxiety-like behavior. Administration of suberoylanilide hydroxamine to neuroLSD1KO mice reactivates egr1 and c-fos transcription and restores the behavioral phenotype. These findings indicate that LSD1 is a molecular transducer of stressful stimuli as well as a stress-response modifier. Indeed, LSD1 expression itself is increased acutely at both the transcriptional and splicing levels by psychosocial stress, suggesting that LSD1 is involved in the adaptive response to stress.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Gillespie syndrome (GLSP) is a rare congenital disorder characterized by partial aniridia, hypotonia, progressive cerebellar hypoplasia, nonprogressive ataxia, and intellectual disability. All ...causative variants to date affect the central or the 3ʹ‐terminal domains of ITPR1 gene and exhibit autosomal recessive or dominant inheritance pattern. We investigated by exome sequencing the molecular cause of GLSP in a family composed by consanguineous healthy parents, two affected siblings and one healthy son. We found the novel splice site variant c.278_279 + 2delACGT located at the 5ʹ‐end of ITPR1. The affected siblings were homozygotes, their parents heterozygous carriers and the variant was absent in the healthy son, indicating a recessive inheritance pattern. The deletion abolished the splice‐donor site at exon 5/intron 5 junction, causing the skipping of exon 5 and the generation of a premature STOP codon. The mutation is predicted to result in the synthesis of a 64‐amino acids nonfunctional protein. The mutant transcript comprised >96% of ITPR1 mRNA in the affected siblings, indicating that a small amount of wild‐type transcript was still present. The novel autosomal recessive mutation here reported is the first variant affecting the ITPR1 N‐terminal suppressor domain, thus extending the spectrum of the pathogenetic variants in GLSP and the range of the associated clinical manifestations.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Identification of predictive molecular alterations in lung adenocarcinoma is essential for accurate therapeutic decisions. Although several molecular approaches are available, a number of issues, ...including tumor heterogeneity, frequent material scarcity, and the large number of loci to be investigated, must be taken into account in selecting the most appropriate technique. MALDI-TOF mass spectrometry (MS), which allows multiplexed genotyping, has been adopted in routine diagnostics as a sensitive, reliable, fast, and cost-effective method. Our aim was to test the reliability of this approach in detecting targetable mutations in non-small cell lung cancer (NSCLC). In addition, we also analyzed low-quality samples, such as cytologic specimens, that often, are the unique source of starting material in lung cancer cases, to test the sensitivity of the system.
We designed a MS-based assay for testing 158 mutations in the EGFR, KRAS, BRAF, ALK, PIK3CA, ERBB2, DDR2, AKT, and MEK1 genes and applied it to 92 NSCLC specimens and 13 liquid biopsies from another subset of NSCLC patients. We also tested the sensitivity of the method to distinguish low represented mutations using serial dilutions of mutated DNA.
Our panel is able to detect the most common NSCLC mutations and the frequency of the mutations observed in our cohort was comparable to literature data. The assay identifies mutated alleles at frequencies of 2.5-10%. In addition, we found that the amount of DNA template was irrelevant to efficiently uncover mutated alleles present at high frequency. However, when using less than 10 ng of DNA, the assay can detect mutations present in at least 10% of the alleles. Finally, using MS and a commercial kit for RT-PCR we tested liquid biopsy from 13 patients with identified mutations in cancers and detected the mutations in 4 (MS) and in 5 samples (RT-PCR).
MS is a powerful method for the routine predictive tests of lung cancer also using low quality and scant tissues. Finally, after appropriate validation and improvement, MS could represent a promising and cost-effective strategy for monitoring the presence and percentage of the mutations also in non-invasive sampling.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Omphalocele is a congenital midline ventral body wall defect that can exist as isolated malformation or as part of a syndrome. It can be considered one of the major and most frequent clinical ...manifestation of Beckwith-Wiedemann Syndrome (BWS) in case of loss of methylation at KCNQ1OT1: Transcription Star Site-Differentially Methylated Region (TSS-DMR) or in presence of CDKN1C mutations. The isolated form of the omphalocele accounts approximately for about the 14% of the total cases and its molecular etiology has never been fully elucidated.
Given the tight relationship with BWS, we hypothesized that the isolated form of the omphalocele could belong to the heterogeneous spectrum of the BWS associated features, representing an endophenotype with a clear genetic connection. We therefore investigated genetic and epigenetic changes affecting BWS imprinted locus at 11p15.5 imprinted region, focusing in particular on the KCNQ1OT1:TSS DMR.
We studied 21 cases of isolated omphalocele detected during pregnancy or at birth and identified the following rare maternally inherited variants: i) the non-coding variant G > A at nucleotide 687 (NR_002728.3) at KCNQ1OT1:TSS-DMR, which alters the methylation pattern of the imprinted allele, in one patient; ii) the deletion c.624-629delGGCCCC at exon 1 of CDKN1C, with unknown clinical significance, in two unrelated cases.
Taken together, these findings suggest that KCNQ1OT1:TSS-DMR could be a susceptibility locus for the isolated omphalocele.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Epigenetic mechanisms play important roles in brain development, orchestrating proliferation, differentiation, and morphogenesis. Lysine‐Specific Demethylase 1 (LSD1 also known as KDM1A and AOF2) is ...a histone modifier involved in transcriptional repression, forming a stable core complex with the corepressors corepressor of REST (CoREST) and histone deacetylases (HDAC1/2). Importantly, in the mammalian CNS, neuronal LSD1‐8a, an alternative splicing isoform of LSD1 including the mini‐exon E8a, sets alongside LSD1 and is capable of enhancing neurite growth and morphogenesis. Here, we describe that the morphogenic properties of neuronal LSD1‐8a require switching off repressive activity and this negative modulation is mediated in vivo by phosphorylation of the Thr369b residue coded by exon E8a. Three‐dimensional crystal structure analysis using a phospho‐mimetic mutant (Thr369bAsp), indicate that phosphorylation affects the residues surrounding the exon E8a‐coded amino acids, causing a local conformational change. We suggest that phosphorylation, without affecting demethylase activity, causes in neurons CoREST and HDAC1/2 corepressors detachment from LSD1‐8a and impairs neuronal LSD1‐8a repressive activity. In neurons, Thr369b phosphorylation is required for morphogenic activity, converting neuronal LSD1‐8a in a dominant‐negative isoform, challenging LSD1‐mediated transcriptional repression on target genes.
LSD1‐8a is a neurospecific splicing isoform of LSD1 that can be phosphorylated at a Thr residue coded by mini‐exon E8a. Phosphorylation enhances LSD1‐8a‐driven neuronal maturation by hampering repressive potential on target morphogenic genes, which is achieved by causing CoREST/HDAC1/2 corepressor complex disassembly. In conclusion, interplay between alternative splicing (AS) and phosphorylation cooperate to set LSD1 repressive potential in the brain.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Alternative splicing in the brain is dynamic and instrumental to adaptive changes in response to stimuli. Lysine-specific demethylase 1 (LSD1/KDM1A) is a ubiquitously expressed histone H3Lys4 ...demethylase that acts as a transcriptional co-repressor in complex with its molecular partners CoREST and HDAC1/2. In mammalian brain, alternative splicing of LSD1 mini-exon E8a gives rise to neuroLSD1, a neurospecific isoform that, upon phosphorylation, acts as a dominant-negative causing disassembly of the co-repressor complex and de-repression of target genes. Here we show that the LSD1/neuroLSD1 ratio changes in response to neuronal activation and such effect is mediated by neurospecific splicing factors NOVA1 and nSR100/SRRM4 together with a novel cis-silencer. Indeed, we found that, in response to epileptogenic stimuli, downregulation of NOVA1 reduces exon E8a splicing and expression of neuroLSD1. Using behavioral and EEG analyses we observed that neuroLSD1-specific null mice are hypoexcitable and display decreased seizure susceptibility. Conversely, in a mouse model of Rett syndrome characterized by hyperexcitability, we measured higher levels of NOVA1 protein and upregulation of neuroLSD1. In conclusion, we propose that, in the brain, correct ratio between LSD1 and neuroLSD1 contributes to excitability and, when altered, could represent a pathogenic event associated with neurological disorders involving altered E/I.
The World Health Organization recently revised the diagnosis of glioma, to integrate molecular parameters, including IDH mutations and codeletion (loss of heterozygosity; LOH) of chromosome arms ...1p/19q, into the definitions of adult glioma histological subtypes. Mutations in the
promoter may also be useful for glioma diagnosis and prognosis. The integration of molecular markers into routine diagnosis requires their rapid and reliable assessment. We propose a MassARRAY (MS)-based test that can identify 1p/19q codeletion using quantitative SNP genotyping and, simultaneously, characterize hotspot mutations in the
,
, and
genes in tumor DNA. We determined the reliability of the MS approach testing 50 gliomas and comparing the MS results with those obtained by standard methods, such as short tandem repeat genotyping, array comparative genomic hybridization (array-CGH) and Fluorescence In Situ Hybridization (FISH) for 1p/19q codeletion and Sanger sequencing for hotspots mutations. The results indicate that MS is suitable for the accurate, rapid, and cost-effective evaluation of chromosome deletions combined with hotspot mutation detection. This MS approach could be similarly exploited in evaluation of LOH in other situations of clinical and/or research importance.
STUDY QUESTION
Is the presence of ESX1 mRNA in seminal fluid (SF) an indicator of residual spermatogenesis in men with non-obstructive azoospermic (NOA)?
SUMMARY ANSWER
ESX1 mRNA in SF is a suitable ...molecular marker for predicting the presence of residual spermatogenesis in testis.
WHAT IS KNOWN ALREADY
ESX1 is an X-linked homeobox gene whose expression in testis is restricted to germ cells. We previously reported, in the testicular biopsies from azoospermic men, a positive correlation between the presence of ESX1 mRNA and residual spermatogenesis.
STUDY DESIGN, SIZE, DURATION
We investigated ESX1 mRNA expression in 70 testicular fragments (TF) and 56 (SF) of 70 NOA men. As controls, we analyzed 8 TF from men with obstructive azoospermic (OA) and 9 SF from normozoospermic men. For all patients we considered the histological classification of testis biopsies and the recovery of spermatozoa by surgical procedures.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Relative ESX1 mRNA expression was evaluated by quantitative RT–PCR using the ΔΔCt method. The results were compared with the recovery of spermatozoa at surgery.
MAIN RESULTS AND THE ROLE OF CHANCE
In TF from NOA patients we found that: (i) ESX1 mRNA level was significantly decreased as the severity of spermatogenic defects increased (P < 0.0001, one-way analysis of variance); (ii) the presence of ESX1 mRNA can predict the success of sperm retrieval (sensitivity: 80%). In SF from NOA patients we found that: (i) ESX1 mRNA was present in 78.5% of NOA men; (ii) the presence of ESX1 mRNA could predict the success of sperm retrieval (sensitivity: 84%).
LIMITATIONS, REASONS FOR CAUTION
Spermatozoa were recovered at surgery in 5 out of 12 patients whose SF was negative for ESX1 mRNA expression. We think that discrepancies between molecular and clinical results could be reduced by analyzing more than one ejaculate from each man.
WIDER IMPLICATIONS OF THE FINDINGS
The data confirm that the ESX1 transcript in the semen of men with NOA is a suitable molecular marker for predicting the presence of residual foci of spermatogenesis in the testis. The implication of these results is that some patients ‘with azoospermia’, although having a severe impairment of spermatogenesis, could still maintain residual foci of spermatogenesis in limited areas of the testes, not always recovered by surgery.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico: Ricerca Corrente grant number RC2014/519-02 to M.M. and from ASM onlus 2010–2011 to M.M. The authors declare that they have no conflict of interest.
X‐linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic ...forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X‐linked heparan sulfate 6‐O‐sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3′‐phosphate, 5′‐phosphosulfate to the sixth position of the N‐sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered “deleterious” by in‐silico tools. An in‐vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
PDF
