Purpose:
To investigate the possible differences between SPECT/CT based whole-remnant and
maximum-voxel dosimetry in patients receiving radio-iodine ablation
treatment of differentiated thyroid ...cancer (DTC).
Methods:
Eighteen DTC patients were administered 1.11 GBq of 131I-NaI after
near-total thyroidectomy and rhTSH stimulation. Two patients had two remnants, so
in total dosimetry was performed for 20 sites. Three SPECT/CT scans
were performed for each patient at 1, 2, and 3–7 days after administration. The
activity, the remnant mass, and the maximum-voxel activity were determined from
these images and from a recovery-coefficient curve derived from
experimental phantom measurements. The cumulated activity was estimated using
trapezoidal-exponential integration. Finally, the absorbed dose was calculated
using S-values for unit-density spheres in whole-remnant
dosimetry and S-values for voxels in
maximum-voxel dosimetry.
Results:
The mean absorbed dose obtained from whole-remnant dosimetry was 40 Gy
(range 2–176 Gy) and from maximum-voxel dosimetry 34 Gy (range 2–145 Gy). For any given
patient, the activity concentrations for each of the three time-points were
approximately the same for the two methods. The effective half-lives varied
(R = 0.865), mainly due to discrepancies in estimation of the
longer effective half-lives. On average, absorbed doses obtained from
whole-remnant dosimetry were 1.2 ± 0.2 (1 SD) higher than for maximum-voxel
dosimetry, mainly due to differences in the
S-values. The method-related differences were however small in
comparison to the wide range of absorbed doses obtained in
patients.
Conclusions:
Simple and consistent procedures for SPECT/CT based whole-volume and maximum-voxel
dosimetry have been described, both based on experimentally
determined recovery coefficients. Generally the results from the two approaches
are consistent, although there is a small, systematic difference in the absorbed
dose
due to differences in the S-values, and some variability due to
differences in the estimated effective half-lives, especially when the effective
half-life is long. Irrespective of the method used, the patient absorbed
doses
obtained span over two orders of magnitude.
Screening for pathogenic variants in the diagnosis of rare genetic diseases can now be performed on all genes thanks to the application of whole exome and genome sequencing (WES, WGS). Yet the ...repertoire of gene-disease associations is not complete. Several computer-based algorithms and databases integrate distinct gene-gene functional networks to accelerate the discovery of gene-disease associations. We hypothesize that the ability of every type of information to extract relevant insights is disease-dependent. We compiled 33 functional networks classified into 13 knowledge categories (KCs) and observed large variability in their ability to recover genes associated with 91 genetic diseases, as measured using efficiency and exclusivity. We developed GLOWgenes, a network-based algorithm that applies random walk with restart to evaluate KCs' ability to recover genes from a given list associated with a phenotype and modulates the prediction of new candidates accordingly. Comparison with other integration strategies and tools shows that our disease-aware approach can boost the discovery of new gene-disease associations, especially for the less obvious ones. KC contribution also varies if obtained using recently discovered genes. Applied to 15 unsolved WES, GLOWgenes proposed three new genes to be involved in the phenotypes of patients with syndromic inherited retinal dystrophies.
DNA variants altering the pre-mRNA splicing process represent an underestimated cause of human genetic diseases. Their association with disease traits should be confirmed using functional assays from ...patient cell lines or alternative models to detect aberrant mRNAs. Long-read sequencing is a suitable technique to identify and quantify mRNA isoforms. Available isoform detection and/or quantification tools are generally designed for the whole transcriptome analysis. However experiments focusing on genes of interest need more precise data fine-tuning and visualization tools.
Here we describe VIsoQLR, an interactive analyzer, viewer and editor for the semi-automated identification and quantification of known and novel isoforms using long-read sequencing data. VIsoQLR is tailored to thoroughly analyze mRNA expression in splicing assays of selected genes. Our tool takes sequences aligned to a reference, and for each gene, it defines consensus splice sites and quantifies isoforms. VIsoQLR introduces features to edit the splice sites through dynamic and interactive graphics and tables, allowing accurate manual curation. Known isoforms detected by other methods can also be imported as references for comparison. A benchmark against two other popular transcriptome-based tools shows VIsoQLR accurate performance on both detection and quantification of isoforms. Here, we present VIsoQLR principles and features and its applicability in a case study example using nanopore-based long-read sequencing. VIsoQLR is available at
https://github.com/TBLabFJD/VIsoQLR
.
The combination of trastuzumab plus pertuzumab plus docetaxel as a first-line therapy in patients with HER2-positive metastatic breast cancer has provided significant clinical benefits compared to ...trastuzumab plus docetaxel alone. However, despite the therapeutic success of existing therapies targeting HER2, tumours invariably relapse. Therefore, there is an urgent need to improve our understanding of the mechanisms governing resistance, so that specific therapeutic strategies can be developed to provide improved efficacy. It is well known that the tumour microenvironment (TME) has a significant impact on cancer behaviour. Cancer-associated fibroblasts (CAFs) are essential components of the tumour stroma that have been linked to acquired therapeutic resistance and poor prognosis in breast cancer. For this reason, it would be of interest to identify novel biomarkers in the tumour stroma that could emerge as therapeutic targets for the modulation of resistant phenotypes. Conditioned medium experiments carried out in our laboratory with CAFs derived from HER2-positive patients showed a significant capacity to promote resistance to trastuzumab plus pertuzumab therapies in two HER2-positive breast cancer cell lines (BCCLs), even in the presence of docetaxel. In order to elucidate the components of the CAF-conditioned medium that may be relevant in the promotion of BCCL resistance, we implemented a multiomics strategy to identify cytokines, transcription factors, kinases and miRNAs in the secretome that have specific targets in cancer cells. The combination of cytokine arrays, label-free LC-MS/MS quantification and miRNA analysis to explore the secretome of CAFs under treatment conditions revealed several up- and downregulated candidates. We discuss the potential role of some of the most interesting candidates in generating resistance in HER2-positive breast cancer.
Highly prevalent respiratory diseases such as asthma and allergy remain a pressing health challenge. Currently, there is an unmet need for precise diagnostic tools capable of predicting the great ...heterogeneity of these illnesses. In a previous study of 94 asthma/respiratory allergy biomarker candidates, we defined a group of potential biomarkers to distinguish clinical phenotypes (i.e. nonallergic asthma, allergic asthma, respiratory allergy without asthma) and disease severity. Here, we analyze our experimental results using complex algorithmic approaches that establish holistic disease models (systems biology), combining these insights with information available in specialized databases developed worldwide. With this approach, we aim to prioritize the most relevant biomarkers according to their specificity and mechanistic implication with molecular motifs of the diseases. The Therapeutic Performance Mapping System (Anaxomics' TPMS technology) was used to generate one mathematical model per disease: allergic asthma (AA), non-allergic asthma (NA), and respiratory allergy (RA), defining specific molecular motifs for each. The relationship of our molecular biomarker candidates and each disease was analyzed by artificial neural networks (ANNs) scores. These analyses prioritized molecular biomarkers specific to the diseases and to particular molecular motifs. As a first step, molecular characterization of the pathophysiological processes of AA defined 16 molecular motifs: 2 specific for AA, 2 shared with RA, and 12 shared with NA. Mechanistic analysis showed 17 proteins that were strongly related to AA. Eleven proteins were associated with RA and 16 proteins with NA. Specificity analysis showed that 12 proteins were specific to AA, 7 were specific to RA, and 2 to NA. Finally, a triggering analysis revealed a relevant role for AKT1, STAT1, and MAPK13 in all three conditions and for TLR4 in asthmatic diseases (AA and NA). In conclusion, this study has enabled us to prioritize biomarkers depending on the functionality associated with each disease and with specific molecular motifs, which could improve the definition and usefulness of new molecular biomarkers.
Mutations in USH2A cause both isolated Retinitis Pigmentosa (RP) and Usher syndrome (that implies RP and hearing impairment). One of the most frequent variants identified in this gene and among these ...patients is the p.(Cys759Phe) change. However, the pathogenic role of this allele has been questioned since it was found in homozygosity in two healthy siblings of a Spanish family. To assess the causative role of USH2A p.(Cys759Phe) in autosomal recessive RP (ARRP) and Usher syndrome type II (USH2) and to establish possible genotype-phenotype correlations associated with p.(Cys759Phe), we performed a comprehensive genetic and clinical study in patients suffering from any of the two above-mentioned diseases and carrying at least one p.(Cys759Phe) allele.
Diagnosis was set according to previously reported protocols. Genetic analyses were performed by using classical molecular and Next-Generation Sequencing approaches. Probands of 57 unrelated families were molecularly studied and 63 patients belonging to these families were phenotypically evaluated.
Molecular analysis characterized 100% of the cases, identifying: 11 homozygous patients for USH2A p.(Cys759Phe), 42 compound heterozygous patients (12 of them with another missense USH2A pathogenic variant and 30 with a truncating USH2A variant), and 4 patients carrying the p.(Cys759Phe) allele and a pathogenic variant in another RP gene (PROM1, CNGB1 or RP1). No additional causative variants were identified in symptomatic homozygous patients. Statistical analysis of clinical differences between zygosity states yielded differences (p≤0.05) in age at diagnosis of RP and hypoacusis, and progression of visual field loss. Homozygosity of p.(Cys759Phe) and compound heterozygosity with another USH2A missense variant is associated with ARRP or ARRP plus late onset hypoacusis (OR = 20.62, CI = 95%, p = 0.041).
The present study supports the role of USH2A p.(Cys759Phe) in ARRP and USH2 pathogenesis, and demonstrates the clinical differences between different zygosity states. Phenotype-genotype correlations may guide the genetic characterization based upon specific clinical signs and may advise on the clinical management and prognosis based upon a specific genotype.
Haploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its ...downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved "PAX6-negative" cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches.
Long-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1.
In both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.
Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the
gene cause Nance-Horan ...syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical
isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced
expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting
, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the
defects.
The HIV reservoir is the main barrier to eradicating HIV infection, and resting memory CD4 T (Trm) cells are one of the most relevant cellular component harboring latent proviruses. This is the first ...study analyzing the transcriptional profile of Trm cells, in two well-characterized groups of HIV patients with distinct mechanisms of viral replication control (spontaneous versus treatment-induced). We use a systems biology approach to unravel subtle but important differences in the molecular mechanisms operating at the cellular level that could be associated with the host’s ability to control virus replication and persistence. Despite the absence of significant differences in the transcriptome of Trm cells between Elite Controllers (ECs) and cART-treated (TX) patients at the single gene level, we found 353 gene ontology (GO) categories upregulated in EC compared with TX. Our results suggest the existence of mechanisms at two different levels: first boosting both adaptive and innate immune responses, and second promoting active viral replication and halting HIV latency in the Trm cell compartment of ECs as compared with TX patients. These differences in the transcriptional profile of Trm cells could be involved in the lower HIV reservoir observed in ECs compared with TX individuals, although mechanistic studies are needed to confirm this hypothesis. Combining transcriptome analysis and systems biology methods is likely to provide important findings to help us in the design of therapeutic strategies aimed at purging the HIV reservoir.
Key messages
HIV-elite controllers have the lowest HIV-DNA content in resting memory CD4 T cells.
HIV-ECs show a particular transcriptional profile in resting memory CD4 T cells.
Molecular mechanisms of enhanced adaptative and innate immune response in HIV-ECs.
High viral replication and low viral latency establishment associate to the EC status.
SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade.
Here we use high resolution ultra-deep sequencing ...to analyze SARS-CoV-2 mutant spectra.
In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon.
We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.
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