25 research outputs found
Carbon nanoparticles as detection labels in antibody microarrays.Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli
No full textThe present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were
used in nucleic acid microarray immunoassays (NAMIAs) for
the detection of di¿erent Shiga toxin-producing Escherichia coli
(STEC) virulence factors: four genes speci¿c for STEC (vt1,vt2,
eae, andehxA) and the gene forE. coli16S (hui). Optimization was
performed using a Box Behnken design, andthe limit of detection
for each virulence factor was established. Finally,this NAMIA using
CNPs was tested with DNA from 48 ¿eld strains originating from
cattle feces, and its performance was evaluated by comparing
results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and speci¿city values higher than 0.80 and
e¿ciency values higher than 0.92. Kappa coe¿cients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference
method used forvt1, eae, and ehxA, and a perfect agreement (k = 1) forvt2 and hui. The excellent agreement between the developed
NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed ¿t for purpose, i.e., it is valuable for fast screening of
ampli¿ed genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.This work was partially supported by the Generalitat Valenciana (BEST/2009/026), the Universidad Politecnica de Valencia (PAID-00-09-2837), and the Dutch Ministry of Agriculture, Nature and Food Quality (Strategic research program Food Safety, Monitoring and Detection KB-06-005). The authors thank Dr. Eva Moller Nielsen at the Danish Veterinary Institute (Copenhagen, Denmark) for providing E. coli control strains and Dr. Lutz Geue (Friedrich-Loeffler-Institut, Wusterhausen, Germany) and Dr. Dorte Dopfer (School of Veterinary Medicine, University of Wisconsin, Madison, WI) for field isolates.Noguera Murray, PS.; Posthuma-Trumpie, GA.; Van Tuil, M.; Van Der Wal, FJ.; De Boer, A.; Moers, APHA.; Van Amerongen, A. (2011). Carbon nanoparticles as detection labels in antibody microarrays.Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli. Analytical Chemistry. 83:8531-8536. https://doi.org/10.1021/ac201823vS853185368
ABC’s of bioabsorption: application of lactide based polymers in fully resorbable cardiovascular stents
No full textAcceptance of Genetic Counseling and Testing in a Hospital‐Based Series of Patients with Gynecological Cancer
No full textImproved Gene Fusion Detection in Childhood Cancer Diagnostics Using RNA Sequencing
No full textPURPOSE: Gene fusions play a significant role in cancer etiology, making their detection crucial for accurate diagnosis, prognosis, and determining therapeutic targets. Current diagnostic methods largely focus on either targeted or low-resolution genome-wide techniques, which may be unable to capture rare events or both fusion partners. We investigate if RNA sequencing can overcome current limitations with traditional diagnostic techniques to identify gene fusion events. METHODS: We first performed RNA sequencing on a validation cohort of 24 samples with a known gene fusion event, after which a prospective pan-pediatric cancer cohort (n = 244) was tested by RNA sequencing in parallel to existing diagnostic procedures. This cohort included hematologic malignancies, tumors of the CNS, solid tumors, and suspected neoplastic samples. All samples were processed in the routine diagnostic workflow and analyzed for gene fusions using standard-of-care methods and RNA sequencing. RESULTS: We identified a clinically relevant gene fusion in 83 of 244 cases in the prospective cohort. Sixty fusions were detected by both routine diagnostic techniques and RNA sequencing, and one fusion was detected only in routine diagnostics, but an additional 24 fusions were detected solely by RNA sequencing. RNA sequencing, therefore, increased the diagnostic yield by 38%-39%. In addition, RNA sequencing identified both gene partners involved in the gene fusion, in contrast to most routine techniques. For two patients, the newly identified fusion by RNA sequencing resulted in treatment with targeted agents. CONCLUSION: We show that RNA sequencing is sufficiently robust for gene fusion detection in routine diagnostics of childhood cancers and can make a difference in treatment decisions
Mutations in GRIP1 Cause Fraser Syndrome
No full textBackground: Fraser syndrome (FS) is a autosomal recessive malformation syndrome characterised by cryptophthalmos, syndactyly and urogenital defects. FS is a genetically heterogeneous condition. Thus far, mutations in FRAS1 and FREM2 have been identified as cause of FS. Both FRAS1 and FREM2 encode extracellular matrix proteins that are essential for the adhesion between epidermal basement membrane and the underlying dermal connective tissues during embryonic development. Mutations in murine Grip1, which encodes a scaffolding protein that interacts with Fras1/Frem proteins, result in FS-like defects in mice. Objective: To test GRIP1 for genetic variants in FS families that do not have mutations in FRAS1 and FREM2. Methods and results: In three unrelated families with parental consanguinity, GRIP1 mutations were found to segregate with the disease in an autosomal recessive manner (donor splice site mutation NM_021150.3:c.2113+1G→C in two families and a 4-bp deletion, NM_021150.3:c.1181_1184del in the third). RT-PCR analysis of the GRIP1 mRNA showed that the c.2113+1G→C splice mutation causes skipping of exon 17, leading to a frame shift and a premature stop of translation. Conclusion: Mutations in GRIP1 cause classic FS in humans
Mutations in GRIP1 cause Fraser syndrome
No full textBackground Fraser syndrome (FS) is a autosomal recessive malformation syndrome characterised by cryptophthalmos, syndactyly and urogenital defects. FS is a genetically heterogeneous condition. Thus far, mutations in FRAS1 and FREM2 have been identified as cause of FS. Both FRAS1 and FREM2 encode extracellular matrix proteins that are essential for the adhesion between epidermal basement membrane and the underlying dermal connective tissues during embryonic development. Mutations in murine Grip1, which encodes a scaffolding protein that interacts with Fras1/Frem proteins, result in FS-like defects in mice. Objective To test GRIP1 for genetic variants in FS families that do not have mutations in FRAS1 and FREM2. Methods and results In three unrelated families with parental consanguinity, GRIP1 mutations were found to segregate with the disease in an autosomal recessive manner (donor splice site mutation NM_021150.3: c.2113+1G -> C in two families and a 4-bp deletion, NM_021150.3: c.1181_1184del in the third). RT-PCR analysis of the GRIP1 mRNA showed that the c.2113+1G -> C splice mutation causes skipping of exon 17, leading to a frame shift and a premature stop of translation. Conclusion Mutations in GRIP1 cause classic FS in human
Carbon Nanoparticles as Detection Labels in Antibody Microarrays. Detection of Genes Encoding Virulence Factors in Shiga Toxin-Producing <i>Escherichia coli</i>
No full textThe present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) and the gene for E. coli 16S (hui). Optimization was performed using a Box–Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays
X-exome sequencing identifies a HDAC8 variant in a large pedigree with X-linked intellectual disability, truncal obesity, gynaecomastia, hypogonadism and unusual face
No full textBACKGROUND: We present a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability, hypogonadism, gynaecomastia, truncal obesity, short stature and recognisable craniofacial manifestations resembling but not identical to Wilson-Turner syndrome. Seven female relatives show a much milder expression of the phenotype. METHODS AND RESULTS: We performed X chromosome exome (X-exome) sequencing in five individuals from this family and identified a novel intronic variant in the histone deacetylase 8 gene (HDAC8), c.164+5G>A, which disturbs the normal splicing of exon 2 resulting in exon skipping, and introduces a premature stop at the beginning of the histone deacetylase catalytic domain. The identified variant completely segregates in this family and was absent in 96 Dutch controls and available databases. Affected female carriers showed a notably skewed X-inactivation pattern in lymphocytes in which the mutated X-chromosome was completely inactivated. CONCLUSIONS: HDAC8 is a member of the protein family of histone deacetylases that play a major role in epigenetic gene silencing during development. HDAC8 specifically controls the patterning of the skull with the mouse HDAC8 knock-out showing craniofacial deformities of the skull. The present family provides the first evidence for involvement of HDAC8 in a syndromic form of intellectual disability
Complex structural variation is prevalent and highly pathogenic in pediatric solid tumors
No full textIn pediatric cancer, structural variants (SVs) and copy-number alterations contribute to cancer initiation as well as progression, thereby aiding diagnosis and treatment stratification. Although suggested to be of importance, the prevalence and biological relevance of complex genomic rearrangements (CGRs) across pediatric solid tumors is largely unexplored. In a cohort of 120 primary tumors, we systematically characterized patterns of extrachromosomal DNA, chromoplexy, and chromothripsis across five pediatric solid cancer types. CGRs were identified in 56 tumors (47%), and in 42 of these tumors, CGRs affect cancer driver genes or result in unfavorable chromosomal alterations. This demonstrates that CGRs are prevalent and pathogenic in pediatric solid tumors and suggests that selection likely contributes to the structural variation landscape. Moreover, carrying CGRs is associated with more adverse clinical events. Our study highlights the potential for CGRs to be incorporated in risk stratification or exploited for targeted treatments
Systematic discovery of gene fusions in pediatric cancer by integrating RNA-seq and WGS
No full textAbstract Background Gene fusions are important cancer drivers in pediatric cancer and their accurate detection is essential for diagnosis and treatment. Clinical decision-making requires high confidence and precision of detection. Recent developments show RNA sequencing (RNA-seq) is promising for genome-wide detection of fusion products but hindered by many false positives that require extensive manual curation and impede discovery of pathogenic fusions. Methods We developed Fusion-sq to overcome existing disadvantages of detecting gene fusions. Fusion-sq integrates and “fuses” evidence from RNA-seq and whole genome sequencing (WGS) using intron–exon gene structure to identify tumor-specific protein coding gene fusions. Fusion-sq was then applied to the data generated from a pediatric pan-cancer cohort of 128 patients by WGS and RNA sequencing. Results In a pediatric pan-cancer cohort of 128 patients, we identified 155 high confidence tumor-specific gene fusions and their underlying structural variants (SVs). This includes all clinically relevant fusions known to be present in this cohort (30 patients). Fusion-sq distinguishes healthy-occurring from tumor-specific fusions and resolves fusions in amplified regions and copy number unstable genomes. A high gene fusion burden is associated with copy number instability. We identified 27 potentially pathogenic fusions involving oncogenes or tumor-suppressor genes characterized by underlying SVs, in some cases leading to expression changes indicative of activating or disruptive effects. Conclusions Our results indicate how clinically relevant and potentially pathogenic gene fusions can be identified and their functional effects investigated by combining WGS and RNA-seq. Integrating RNA fusion predictions with underlying SVs advances fusion detection beyond extensive manual filtering. Taken together, we developed a method for identifying candidate gene fusions that is suitable for precision oncology applications. Our method provides multi-omics evidence for assessing the pathogenicity of tumor-specific gene fusions for future clinical decision making
