| 1. |
- Adolfsson, Emma, 1977-, et al.
(författare)
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CNV-Z; a new tool for detecting copy number variation in next generation sequencing data
- 2023
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Ingår i: SoftwareX. - : Elsevier. - 2352-7110. ; 24
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Tidskriftsartikel (refereegranskat)abstract
- We developed an efficient approach to diagnostic copy number analysis of targeted gene panel or whole exome sequence (WES) data. Here we present CNV-Z as a new tool for detection of copy number variants (CNVs). Deletions and duplications of chromosomal regions are widely implicated in both genomic evolution and genetic disorders. However, calling CNVs from targeted or exome sequence data is challenging. In most cases, the copy number of a chromosomal region is estimated as the depth of reads mapping to a certain bin or sliding window divided by the expected number of reads derived from a set of reference samples. This approach will inevitably miss smaller CNVs on an irregular basis, and quite frequently results in a disturbing number of false positive CNVs. We developed an alternative approach to detect CNVs based on deviation from expected read depth per position, instead of region. Cautiously used, the cohort of samples in the same run will do as a reference. With appropriate filtering, given high quality DNA and a set of suitable reference samples, CNV-Z detects CNVs ranging in length from one nucleotide to an entire chromosome, with few false positives. Performance is proved by benchmarking using both in-house targeted gene panel NGS data and a publicly available NGS dataset, both sets with multiplex ligation-dependent amplification probe (MLPA) validated CNVs. The outcome shows that CNV-Z detects single- and multi-exonic CNVs with high specificity and sensitivity using different kind of NGS data. On gene level, CNV-Z shows both excellent sensitivity and specificity. Compared to competing CNV callers, CNV-Z shows higher specificity and positive predictive value for detecting exonic CNVs.
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| 2. |
- Adolfsson, Emma, 1977-, et al.
(författare)
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Technical in-depth comparison of two massive parallel DNA-sequencing methods for formalin-fixed paraffin-embedded tissue from victims of sudden cardiac death
- 2021
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Ingår i: Forensic Science International. - : Elsevier. - 1872-4973 .- 1878-0326. ; 53
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Tidskriftsartikel (refereegranskat)abstract
- Sudden cardiac death (SCD) is a tragic and traumatic event. SCD is often associated with hereditary genetic disease and in such cases, sequencing of stored formalin fixed paraffin embedded (FFPE) tissue is often crucial in trying to find a causal genetic variant. This study was designed to compare two massive parallel sequencing assays for differences in sensitivity and precision regarding variants related to SCD in FFPE material. From eight cases of SCD where DNA from blood had been sequenced using HaloPlex, corresponding FFPE samples were collected six years later. DNA from FFPE samples were amplified using HaloPlex HS, sequenced on MiSeq, representing the first method, as well as amplified using modified Twist and sequenced on NextSeq, representing the second method. Molecular barcodes were included to distinguish artefacts from true variants. In both approaches, read coverage, uniformity and variant detection were compared using genomic DNA isolated from blood and corresponding FFPE tissue, respectively. In terms of coverage uniformity, Twist performed better than HaloPlex HS for FFPE samples. Despite higher overall coverage, amplicon-based HaloPlex technologies, both for blood and FFPE tissue, suffered from design and/or performance issues resulting in genes lacking complete coverage. Although Twist had considerably lower overall mean coverage, high uniformity resulted in equal or higher fraction of genes covered at ≥ 20X. By comparing variants found in the matched samples in a pre-defined cardiodiagnostic gene panel, HaloPlex HS for FFPE material resulted in high sensitivity, 98.0% (range 96.6-100%), and high precision, 99.9% (range 99.5-100%) for moderately fragmented samples, but suffered from reduced sensitivity (range 74.2-91.1%) in more severely fragmented samples due to lack of coverage. Twist had high sensitivity, 97.8% (range 96.8-98.7%) and high precision, 99.9% (range 99.3-100%) in all analyzed samples, including the severely fragmented samples.
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| 3. |
- Green, Anna, 1973-, et al.
(författare)
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Copy number variants in familial hypercholesterolemia genes using targeted NGS, validated through optical genome mapping
- 2024
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Ingår i: European Journal of Human Genetics. - : Nature Portfolio. - 1018-4813 .- 1476-5438. ; 32:Suppl. 1, s. 159-159
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Background/Objectives: Familial hypercholesterolemia (FH) is a common genetic disorder which is primarily caused by pathogenic variants in the LDLR, APOB, and PCSK9 genes. Approximately 10% of pathogenic variants in LDLR may be CNVs. Here, we combine NGS, MLPA, and Optical Genome Mapping (OGM) to investigate CNVs in LDLR.Methods: A NGS panel was designed for whole gene sequencing (8 genes) of 100 FH patients using Twist technology and Illumina platform. CNVs were detected using CNVexpo, and an in-house pipeline for base-resolved normalized coverage. Identified CNVs were validated using MLPA and OGM. Bionano Services Lab performed the OGM procedure. Purified gDNA was labeled using Direct Label and Stain DNA Labeling Kit. Saphyr chip was run aiming for 100X coverage. De novo assembly and Variant Annotation pipelines were executed on Bionano Solve v3.7. Bionano Access v1.7 was used for CNV reporting and visualization.Results: In five out of 100 samples NGS and MLPA data showed heterozygous deletions in LDLR. Three deletions, affecting different exons, was analyzed and confirmed using OGM. In two samples, OGM better defined the breakpoints as well as the size of the event, which expanded far beyond the gene of interest. In one sample, an additional CNV of SLCO1B1, a pharmaco-gene, important for transport of statins used for FH treatment was identified.Conclusion: CNVs in FH genes in FH patients could be detected using targeted NGS, which was further confirmed by MLPA and characterized using OGM.
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| 4. |
- Gréen, Anna, 1973-, et al.
(författare)
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Histone H1 interphase phosphorylation pattern becomes largely established during G1/S transition in proliferating cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Histone H1 is an important constituent of chromatin, and is believed to be involved in regulation of chromatin structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase and again decondensed before re-entry into G1. This has been connected to increasing phosphorylation of H1 histones during the cell cycle. However, many of these experiments have been performed in non-human and human cancer cell lines, and by the use of cell synchronization techniques and cell cycle-arresting drugs. In this study, we have investigated the H1 subtype composition and phosphorylation pattern in the cell cycle. Exponentially growing normal human activated T cells and Jurkat lymphoblastoid cells were sorted by fluorescence activated cell sorting into G1, S and G2/M populations, without the use of cell cycle arresting drugs. We found that the H1.5 protein level increased after T-cell activation. Our data indicate that serine phosphorylation of H1 subtypes occurred to a large extent in late G1 phase or early S, while some additional serine phosphorylation took place during S, G2 and M phases. Furthermore, our data suggest that the newly synthesized H1 molecules during S phase also achieve a similar phosphorylation pattern as the previous ones. Jurkat cells showed more extended H1.5 phosphorylation in G1 compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G1 in Jurkat cells. In conclusion, our data is consistent with a model where a major part of interphase H1 serine phosphorylation takes place within a narrow time window during the G1/Stransition. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication.
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| 5. |
- Koskela, Anita, 1979-, et al.
(författare)
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Evaluation of Microsatellite instability score from GMS560 DNA panel
- 2022
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Microsatellite instability is characterised by gains or losses of nucleotides in short tandem repeat sequences, microsatellites, dispersed throughout the human genome. Microsatellite instability status is a molecular fingerprint for DNA mismatch repair deficiency. Clinical detection of microsatellite instability status is important for identifying inherited disease in patients with colorectal and endometrial cancer but has also a prognostic value for survival and prediction of treatment response. Lately, microsatellite instability has been used as a tumor agnostic biomarker that predicts response to immune checkpoint inhibitors. To identify microsatellite instability status clinically, PCR and immunohistochemistry have been the gold standard. On the contrary, next generation sequencing provide simultaneous accession of large number of microsatellite loci and can be combined with detection of several other biomarkers. The national collaboration Genome Medicine Sweden have developed a solid tumour gene panel composed of 560 cancer associated genes with integrated microsatellite instability score. Our aim was to validate the microsatellite instability status based on microsatellite instability score from GMS560 DNA panel against the clinically used methods. Extracted DNA (100 ng) from formalin fixed paraffin embedded tissue sections with various tumour cell content >10% were analysed. During target enrichment sequencing analysis, allelic distribution from 5000 microsatellite markers were calculated by MSIsensor Pro to generate an instability score. The cohort consisted of microsatellite instable verified colorectal cancer samples (n=20), microsatellite stable solid tumour material (n=60). Preliminary results generated a microsatellite instability score for the colorectal cancer samples with a mean of 26.5 % (CI: 23.4-29.6, range: 16.9-32.3). Microsatellite stable tumour samples had a mean microsatellite instability score of 1.5 % (CI: 0.93-2.07, range: 1-4.45). In conclusion, we found the microsatellite instability score from GMS560 DNA panel to be both diagnostically sensitive and specific for determining MSI status due to obvious separation in instability.
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| 6. |
- Olofsson, Emma, 1981-, et al.
(författare)
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Whole exome sequencing of FFPE samples-expanding the horizon of forensic molecular autopsies
- 2023
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Ingår i: International journal of legal medicine. - : Springer. - 0937-9827 .- 1437-1596. ; 137:4, s. 1215-1234
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Tidskriftsartikel (refereegranskat)abstract
- Forensic molecular autopsies have emerged as a tool for medical examiners to establish the cause of death. It is particularly useful in sudden unexplained deaths where the cause of death cannot be determined with a regular medical autopsy. We provide the first study of exome data from formalin-fixed paraffin-embedded samples (FFPE) paired with data from high-quality blood samples in forensic applications. The approach allows exploration of the potential to use FFPE samples for molecular autopsies and identify variants in extensive exome data. We leverage the high uniformity of the hybridization capture approach provided by Twist Bioscience to target the complete exome and sequence the libraries on a NextSeq 550. Our findings suggest that exome sequencing is feasible for 24 out of a total of 35 included FFPE samples. When successful, the coverage across the exome is comparatively high (> 90% covered to 20X) and uniform (fold80 below 1.5). Detailed variant comparisons for matched FFPE and blood samples show high concordance with few false variants (positive predictive value of 0.98 and a sensitivity of 0.97) with no distinct FFPE artefacts. Ultimately, we apply carefully constructed forensic gene panels in a stepwise manner to find genetic variants associated with the clinical phenotype and with relevance to the sudden unexplained death.
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| 7. |
- Sandestig, Anna, et al.
(författare)
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Could Dissimilar Phenotypic Effects of ACTB Missense Mutations Reflect the Actin Conformational Change? : Two Novel Mutations and Literature Review
- 2018
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Ingår i: Molecular Syndromology. - : S. Karger. - 1661-8769 .- 1661-8777. ; 9:5, s. 259-265
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Tidskriftsartikel (refereegranskat)abstract
- The beta-actin gene encodes 1 of 6 different actin proteins. De novo heterozygous missense mutations in ACTB have been identified in patients with Baraitser-Winter syndrome (BRWS) and also in patients with developmental disorders other than BRWS, such as deafness, dystonia, and neutrophil dysfunction. We describe 2 different novel de novo missense ACTB mutations, c.208Camp;gt;G (p.Pro70Ala) and c.511Camp;gt;T (p.Leu171Phe), found by trio exome sequencing analysis of 2 unrelated patients: an 8-year-old boy with a suspected BRWS and a 4-year-old girl with unclear developmental disorder. The mutated residue in the first case is situated in the actin H-loop, which is involved in actin polymerization. The mutated residue in the second case (p.Leu171Phe) is found at the actin barbed end in the W-loop, important for binding to profilin and other actin-binding molecules. While the boy presented with a typical BRWS facial appearance, the girl showed facial features not recognizable as a BRWS gestalt as well as ventricular arrhythmia, cleft palate, thrombocytopenia, and gray matter heterotopia. We reviewed previously published ACTB missense mutations and ascertained that a number of them do not cause typical BRWS. By comparing clinical and molecular data, we speculate that the phenotypic differences found in ACTB missense mutation carriers might supposedly be dependent on the conformational change of ACTB.
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| 8. |
- Engström, Karolina, et al.
(författare)
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Novel SMAD3 p.Arg386Thr genetic variant co-segregating with thoracic aortic aneurysm and dissection
- 2020
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Ingår i: Molecular Genetics & Genomic Medicine. - : John Wiley & Sons. - 2324-9269. ; 8:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Pathogenic variants in the SMAD3 gene affecting the TGF-beta/SMAD3 signaling pathway with aortic vessel involvement cause Loeys-Dietz syndrome 3, also known as aneurysms-osteoarthritis syndrome.Methods: Description of clinical history of a family in Sweden using clinical data, DNA sequencing, bioinformatics, and pedigree analysis.Results: We report a novel SMAD3 variant, initially classified as a genetic variant of uncertain clinical significance (VUS), and later found to be co-segregating with aortic dissection in the family. The index patient presented with a dissecting aneurysm of the aorta including the ascending, descending, and abdominal parts. Genotype analysis revealed a heterozygous missense SMAD3 variant: NM_005902.3(SMAD3): c.11576G > C (p.Arg386Thr). The same variant was also identified in a 30 years old formalin-fixed paraffin-embedded block of tissue from a second cousin, who died at 26 years of age from a dissecting aneurysm of the aorta.Conclusion: A "variant of uncertain significance" according to the ACMG guidelines has always a scope for reappraisal. Genetic counselling to relatives, and the offering of surveillance service is important to families with aortic aneurysm disease. The report also highlight the potential use of FFPE analysis from deceased relatives to help in the interpretation of variants.
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| 9. |
- Garcia-Cordero, Indira, et al.
(författare)
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Functional connectivity changes in neurodegenerative biomarker-positive athletes with repeated concussions
- 2024
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Ingår i: JOURNAL OF NEUROLOGY. - 0340-5354 .- 1432-1459.
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Tidskriftsartikel (refereegranskat)abstract
- Multimodal biomarkers may identify former contact sports athletes with repeated concussions and at risk for dementia. Our study aims to investigate whether biomarker evidence of neurodegeneration in former professional athletes with repetitive concussions (ExPro) is associated with worse cognition and mood/behavior, brain atrophy, and altered functional connectivity. Forty-one contact sports athletes with repeated concussions were divided into neurodegenerative biomarker-positive (n = 16) and biomarker-negative (n = 25) groups based on positivity of serum neurofilament light-chain. Six healthy controls (negative for biomarkers) with no history of concussions were also analyzed. We calculated cognitive and mood/behavior composite scores from neuropsychological assessments. Gray matter volume maps and functional connectivity of the default mode, salience, and frontoparietal networks were compared between groups using ANCOVAs, controlling for age, and total intracranial volume. The association between the connectivity networks and sports characteristics was analyzed by multiple regression analysis in all ExPro. Participants presented normal-range mean performance in executive function, memory, and mood/behavior tests. The ExPro groups did not differ in professional years played, age at first participation in contact sports, and number of concussions. There were no differences in gray matter volume between groups. The neurodegenerative biomarker-positive group had lower connectivity in the default mode network (DMN) compared to the healthy controls and the neurodegenerative biomarker-negative group. DMN disconnection was associated with increased number of concussions in all ExPro. Biomarkers of neurodegeneration may be useful to detect athletes that are still cognitively normal, but with functional connectivity alterations after concussions and at risk of dementia.
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| 10. |
- Gavanas, Anna, 1973, et al.
(författare)
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Skype-mödrar och priset för rena hem
- 2013
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Ingår i: Arena. - 1652-0556. ; :2, s. 26-27
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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