Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the ...genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.
Context: Poorly differentiated carcinomas represent an aggressive group of thyroid tumors with controversial classification placement and poorly understood pathogenesis. Molecular data in this group ...of tumors are extremely heterogeneous, possibly reflecting different inclusion criteria. Recently homogeneous diagnostic criteria have been proposed by our group (Turin proposal) that need to be complemented by detailed molecular characterization.
Objective: The objective of the study was to define a comprehensive molecular typing of poorly differentiated thyroid carcinomas classified following homogeneous diagnostic criteria.
Design: Sixty-five cases of poorly differentiated carcinoma selected following the Turin proposal have been screened for N-, K-, H-RAS, BRAF, RET/PTC1 and 3, and PAX8/PPARγ mutations-rearrangements using alternative techniques and in two different laboratories. Molecular data were compared with clinical pathological parameters and survival by univariate and multivariate analysis.
Results: RAS mutations in codon 61 were by far the most common genetic alteration in poorly differentiated carcinomas (23% of cases), with all mutation in NRAS except one in the HRAS gene. A single BRAF mutation was found in a poorly differentiated carcinoma with a residual component of a tall cell variant of papillary carcinoma. No KRAS, RET/PTC, or PAX8/PPARγ genetic alteration was detected. In this series, the presence of RAS mutations was a unique negative prognostic parameter at multivariate analysis.
Conclusions: The present study demonstrates that strictly classified poorly differentiated carcinomas are genetically homogeneous, RAS mutations being the almost exclusive genetic event. Moreover, the detection of RAS mutations might be clinically relevant for the prognostic stratification of these tumors.
RAS mutations are the predominant molecular event in poorly differentiated thyroid carcinoma when WHO criteria for classification are strictly applied and represent a strong indicator of poor prognosis.
Primary Hyperoxaluria type I (PH1) is a rare disease caused by mutations in the AGXT gene encoding alanine:glyoxylate aminotransferase (AGT), a liver enzyme involved in the detoxification of ...glyoxylate, the failure of which results in accumulation of oxalate and kidney stones formation. The role of protein misfolding in the AGT deficit caused by most PH1-causing mutations is increasingly being recognized. In addition, the genetic background in which a mutation occurs is emerging as a critical risk factor for disease onset and/or severity. Based on these premises, in this study we have analyzed the clinical, biochemical and cellular effects of the p.Ile56Asn mutation, recently described in a PH1 patient, as a function of the residue at position 11, a hot-spot for both polymorphic (p.Pro11Leu) and pathogenic (p.Pro11Arg) mutations. We have found that the p.Ile56Asn mutation induces a structural defect mostly related to the apo-form of AGT. The effects are more pronounced when the substitution of Ile56 is combined with the p.Pro11Leu and, at higher degree, the p.Pro11Arg mutation. As compared with the non-pathogenic forms, AGT variants display reduced expression and activity in mammalian cells. Vitamin B6, a currently approved treatment for PH1, can overcome the effects of the p.Ile56Asn mutation only when it is associated with Pro at position 11. Our results provide a first proof that the genetic background influences the effects of PH1-causing mutations and the responsiveness to treatment and suggest that molecular and cellular studies can integrate clinical data to identify the best therapeutic strategy for PH1 patients.
•AGT misfolding is one of the main pathogenic mechanisms underlying PH1•PH1 onset and/or severity is influenced by the AGXT genetic background•The I56N mutation causes a folding defect in apoAGT•The effects of the Ile56 mutation are influenced by substitutions at Pro11•Molecular and cellular data on AGT variants correlate with the clinical phenotype and response to treatment of PH1 patients
Genetic testing availability in the health care system is rapidly increasing, along with the diffusion of next-generation sequencing (NGS) into diagnostics. These issues make imperative the ...knowledge-drive optimization of testing in the clinical setting. Time estimations of wet laboratory procedure in Italian molecular laboratories offering genetic diagnosis were evaluated to provide data suitable to adjust efficiency and optimize health policies and costs. A survey was undertaken by the Italian Society of Human Genetics (SIGU). Forty-two laboratories participated. For most molecular techniques, the most time-consuming steps are those requiring an intensive manual intervention or in which the human bias can affect the global process time-performances. For NGS, for which the study surveyed also the interpretation time, the latter represented the step that requiring longer times. We report the first survey describing the hands-on times requested for different molecular diagnostics procedures, including NGS. The analysis of this survey suggests the need of some improvements to optimize some analytical processes, such as the implementation of laboratory information management systems to minimize manual procedures in pre-analytical steps which may affect accuracy that represents the major challenge to be faced in the future setting of molecular genetics laboratory.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was ...efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand beta-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) gamma strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by beta-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor beta, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4(+) T cells, IL-1beta, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.
Purpose: XRCC1 and XPD play key roles in the repair of DNA lesions and adducts. Contrasting findings have been reported on the effect
of polymorphisms of these genes on the response to platinum-based ...chemotherapy in advanced non–small-cell lung cancer (NSCLC).
This study aimed to investigate the relationship between the XPD Lys751Gln and XRCC1 Arg399Gln genotypes and outcome in lung cancer patients.
Experimental Design: We genotyped 203 NSCLC and 45 small-cell lung carcinoma (SCLC) patients for the two polymorphisms. Most of the patients (81%)
received a platinum-based chemotherapy.
Results: The patients' genotype frequencies did not significantly differ from controls and both groups were in Hardy-Weinberg equilibrium
for the two polymorphisms. The XRCC1 399 Gln/Gln variant genotype was associated with a higher median survival time (80 weeks versus 54.6 weeks for the Arg/Gln
heterozygous and 55.6 weeks for the wild-type Arg/Arg genotype; P = 0.09). At the multivariable analysis adjusted for histology, stage of the disease, performance status, age, and gender,
the Gln/Gln genotype was associated with a better survival of borderline significance in the subgroup of patients treated
with cisplatin (hazard ratio, 0.55; 95% CI, 0.30-1.00); this association became significant for those with grade 3-4 clinical
toxicity (hazard ratio, 0.46; 95% CI, 0.22-0.98). No association between XPD Lys751Gln genotype and clinical outcome was found.
Conclusion: This prospective investigation provides suggestive evidence of a favorable effect of the XRCC1 399 Gln/Gln genotype on survival in platinum-treated NSCLC and, for the first time, in SCLC patients also. This contrasts
with other authors who did not include non–platinum-treated patients, but it does fit the expectation for a suboptimal ability
to remove DNA adducts.
A high prevalence of atrial fibrillation/atrial flutter (AF/AFl) has been reported in small series of Brugada patients, with discordant data.
The purpose of this study was to analyze, in a large ...population of Brugada patients, the prevalence of AF/AFl, its correlation with prognosis, and the efficacy of hydroquinidine (HQ) treatment.
Among 560 patients with Brugada type 1 ECG (BrECG), 48 (9%) had AF/AFl. Three groups were considered: 23 patients with BrECG pattern recognized before AF/AFl (group 1); 25 patients first diagnosed with AF/AFl in whom Class IC antiarrhythmic drugs administered for cardioversion/prophylaxis unmasked BrECG (group 2); and 512 patients without AF/AFl (group 3). Recurrence of AF/AFl and occurrence of ventricular arrhythmias were evaluated at follow-up.
Mean age was 47 ± 15 years, 59 ± 11 years, and 44 ± 14 years in groups 1, 2, and 3, respectively. Seven subjects (32%) in group 1 had syncope/aborted sudden death, 1 (4%) in group 2, and 122 (24%) in group 3. Ventricular arrhythmia occurred in three patients in group 1, none in group 2, and 10 in group 3 at median follow-up of 51, 68, and 41 months, respectively. Nine patients in group 1 and nine in group 2 received HQ for AF/AFl prophylaxis; on therapy, none had AF/AFl recurrence.
Prevalence of AF/AFl in Brugada patients is higher than in the general population of the same age. Patients in group 1 are younger than those in group 2 and have a worse prognosis compared to both groups 2 and 3. HQ therapy has proved useful and safe in patients with AF/AFl and BrECG.
Brugada syndrome (BrS) is marked by coved ST-segment elevation and increased risk of sudden cardiac death. The genetics of this syndrome are elusive in over half of the cases. Variants in the
gene ...are the single most common known genetic unifier, accounting for about a third of cases. Research models, such as animal models and cell lines, are limited. In the present study, we report the novel NM_198056.2:c.1111C>T (p.Gln371*) heterozygous variant in the
gene, as well as its segregation with BrS in a large family. The results herein suggest a pathogenic effect of this variant. Functional studies are certainly warranted to characterize the molecular effects of this variant.
Patients with adrenal incidentalomas (AI) may experience detrimental consequences due to a minimal cortisol excess sustained by adrenal adenoma. SNPs of the glucocorticoid receptor gene (NR3C1) ...modulate individual sensitivity to glucocorticoids and may interfere with the clinical presentation.
To compare the frequency of N363S, ER22/23EK and BclI SNPs in patients with AI with the general population and to evaluate whether these SNPs are linked to consequences of cortisol excess.
Multicentric, retrospective analysis of patients referred from 2010 to 2014 to 4 centers (Orbassano, Milano, Messina Italy and Zagreb Croatia).
411 patients with AI; 153 males and 258 females and 186 from blood donors.
All patients and controls were genotyped for BclI, N363S and ER22/23EK and SNPs frequency was associated with clinical and hormonal features.
SNP frequency was: SNP frequency was: N363S 5.4% (MAF 0.027), BclI 54.7% (MAF 0.328), ER22/23EK 4.4% (MAF 0.022), without any significant difference between patients and controls. N363S was more frequent in hypertensive patients (p = 0.03) and was associated with hypertension (p = 0.015) in patients with suppressed cortisol after the 1-mg DST.
Our results demonstrate that SNPs of the glucocorticoid receptor gene do not play a pathogenetic role for AI. The impact of any single SNP on the phenotypic expression of minimal cortisol excess is limited and their analysis does not provide additional data that may be exploited for patient management.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Genomic structural variants (SVs) can affect many genes and regulatory elements. Therefore, the molecular mechanisms driving the phenotypes of patients carrying de novo SVs are frequently unknown.
We ...applied a combination of systematic experimental and bioinformatic methods to improve the molecular diagnosis of 39 patients with multiple congenital abnormalities and/or intellectual disability harboring apparent de novo SVs, most with an inconclusive diagnosis after regular genetic testing.
In 7 of these cases (18%), whole-genome sequencing analysis revealed disease-relevant complexities of the SVs missed in routine microarray-based analyses. We developed a computational tool to predict the effects on genes directly affected by SVs and on genes indirectly affected likely due to the changes in chromatin organization and impact on regulatory mechanisms. By combining these functional predictions with extensive phenotype information, candidate driver genes were identified in 16/39 (41%) patients. In 8 cases, evidence was found for the involvement of multiple candidate drivers contributing to different parts of the phenotypes. Subsequently, we applied this computational method to two cohorts containing a total of 379 patients with previously detected and classified de novo SVs and identified candidate driver genes in 189 cases (50%), including 40 cases whose SVs were previously not classified as pathogenic. Pathogenic position effects were predicted in 28% of all studied cases with balanced SVs and in 11% of the cases with copy number variants.
These results demonstrate an integrated computational and experimental approach to predict driver genes based on analyses of WGS data with phenotype association and chromatin organization datasets. These analyses nominate new pathogenic loci and have strong potential to improve the molecular diagnosis of patients with de novo SVs.
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