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Eisfeldt, J., Lundin, J., Pettersson, M., Kvarnung, M., Lieden, A., Sahlin, E., . . . Lindstrand, A. (2019). From cytogenetics to cytogenomics whole genome sequencing as a comprehensive genetic test in rare disease diagnostics. Molecular Cytogenetics, 12
Open this publication in new window or tab >>From cytogenetics to cytogenomics whole genome sequencing as a comprehensive genetic test in rare disease diagnostics
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2019 (English)In: Molecular Cytogenetics, ISSN 1755-8166, E-ISSN 1755-8166, Vol. 12Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
BMC, 2019
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-255379 (URN)000474609600033 ()
Note

QC 20190731

Available from: 2019-07-31 Created: 2019-07-31 Last updated: 2019-07-31Bibliographically approved
Nilsson, D., Eisfeldt, J., Lundin, J., Pettersson, M., Kvarnung, M., Lieden, A., . . . Lindstrand, A. (2019). From cytogenetics to cytogenomics: whole genome sequencing as a comprehensive genetic test in rare disease diagnostics. Paper presented at 52nd Conference of the European-Society-of-Human-Genetics (ESHG), JUN 15-18, 2019, Gothenburg, SWEDEN. European Journal of Human Genetics, 27, 1666-1667
Open this publication in new window or tab >>From cytogenetics to cytogenomics: whole genome sequencing as a comprehensive genetic test in rare disease diagnostics
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2019 (English)In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 27, p. 1666-1667Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Rare genetic diseases are caused by different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements. Recent data indicates that whole genome sequencing (WGS) may be used as a comprehensive test to identify multiple types of pathologic genetic aberrations in a single analysis.

We present FindSV, a bioinformatic pipeline for detection of balanced (inversions and translocations) and unbalanced (deletions and duplications) structural variants (SVs). First, FindSV was tested on 106 validated deletions and duplications with a median size of 850 kb (min: 511 bp, max: 155 Mb). All variants were detected. Second, we demonstrated the clinical utility in 138 monogenic WGS panels. SV analysis yielded 11 diagnostic findings (8%). Remarkably, a complex structural rearrangement involving two clustered deletions disrupting SCN1A, SCN2A, and SCN3A was identified in a three months old girl with epileptic encephalopathy. Finally, 100 consecutive samples referred for clinical microarray were also analyzed by WGS. The WGS data was screened for large (>2 kbp) SVs genome wide, processed for visualization in our clinical routine arrayCGH workflow with the newly developed tool vcf2cytosure, and for exonic SVs and SNVs in a panel of 700 genes linked to intellectual disability. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. The diagnostic rate (29%) was doubled compared to clinical microarray (12%).

In conclusion, using WGS we have detected a wide range of structural variation with high accuracy, confirming it a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-262966 (URN)10.1038/s41431-019-0494-2 (DOI)000489313905114 ()2-s2.0-85073110688 (Scopus ID)
Conference
52nd Conference of the European-Society-of-Human-Genetics (ESHG), JUN 15-18, 2019, Gothenburg, SWEDEN
Note

QC 20191104

Available from: 2019-11-04 Created: 2019-11-04 Last updated: 2019-11-04Bibliographically approved
Lindstrand, A., Eisfeldt, J., Pettersson, M., Carvalho, C. M. B., Kvarnung, M., Grigelioniene, G., . . . Nilsson, D. (2019). From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability. Genome Medicine, 11(1), Article ID 68.
Open this publication in new window or tab >>From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability
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2019 (English)In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 11, no 1, article id 68Article in journal (Refereed) Published
Abstract [en]

BackgroundSince different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test.MethodsWe analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n=68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n=156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure.ResultsFirst, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850kb (min 500bp, max 155Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (>10kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data.Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively.ConclusionThe overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

Place, publisher, year, edition, pages
BMC, 2019
Keywords
Whole-genome sequencing, Intellectual disability, Monogenic disease, Copy number variation, Structural variation, Single nucleotide variant, Uniparental disomy, Repeat expansion
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-264890 (URN)10.1186/s13073-019-0675-1 (DOI)000495667900001 ()31694722 (PubMedID)2-s2.0-85074626429 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20191218

Available from: 2019-12-18 Created: 2019-12-18 Last updated: 2020-01-09Bibliographically approved
Alagaratnam, S., Pedersen, G. M., McAdam, S., Wirta, V., Lundeberg, J., Duno, M., . . . Undlien, D. (2019). Quality improvement in clinical NGS through a peer-driven Nordic collaboration. Paper presented at 52nd Conference of the European-Society-of-Human-Genetics (ESHG), JUN 15-18, 2019, Gothenburg, SWEDEN. European Journal of Human Genetics, 27, 1622-1623
Open this publication in new window or tab >>Quality improvement in clinical NGS through a peer-driven Nordic collaboration
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2019 (English)In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 27, p. 1622-1623Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-262967 (URN)000489313905023 ()
Conference
52nd Conference of the European-Society-of-Human-Genetics (ESHG), JUN 15-18, 2019, Gothenburg, SWEDEN
Note

QC 20191129

Available from: 2019-11-29 Created: 2019-11-29 Last updated: 2019-11-29Bibliographically approved
Lundin, K. E., Wang, Q., Hamasy, A., Marits, P., Uzunel, M., Wirta, V., . . . Smith, C. I. (2018). Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report. BMC Pediatrics, 18, Article ID 285.
Open this publication in new window or tab >>Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report
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2018 (English)In: BMC Pediatrics, ISSN 1471-2431, E-ISSN 1471-2431, Vol. 18, article id 285Article in journal (Refereed) Published
Abstract [en]

Background: A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 +3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. Case presentation: We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naive CD4(+) and CD8(+) T-cells, increased number of activated effector memory CD8(+) T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient's cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. Conclusions: There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing.

Place, publisher, year, edition, pages
Springer, 2018
Keywords
Congenital disorder of glycosylation, Hyper-IgE, N-acetylglucosamine-phosphate mutase, PGM3 enzyme activity, Phosphoglucomutase 3, Primary immunodeficiency, Splice-modifying mutation
National Category
Hematology
Identifiers
urn:nbn:se:kth:diva-234589 (URN)10.1186/s12887-018-1258-9 (DOI)000443127600001 ()30157810 (PubMedID)2-s2.0-85052577427 (Scopus ID)
Funder
Swedish Cancer Society, CAN2013/389The Swedish Medical Association, K2015-68X-11247-21-3Swedish Association of Local Authorities and Regions, ALF-projects 2012006, ALFGBG-438361and ALFGBG-672451
Note

QC 20180914

Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2018-09-14Bibliographically approved
Nazaryan-Petersen, L., Eisfeldt, J., Pettersson, M., Lundin, J., Nilsson, D., Wincent, J., . . . Lindstrand, A. (2018). Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization. PLoS Genetics, 14(11), Article ID e1007780.
Open this publication in new window or tab >>Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization
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2018 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 11, article id e1007780Article in journal (Refereed) Published
Abstract [en]

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

Place, publisher, year, edition, pages
Public Library of Science, 2018
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-240782 (URN)10.1371/journal.pgen.1007780 (DOI)000452454300037 ()30419018 (PubMedID)2-s2.0-85057212366 (Scopus ID)
Funder
Swedish Research Council, 2013-2603 2017-02936Marianne and Marcus Wallenberg Foundation, 2014.0084Swedish Society for Medical Research (SSMF)Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20190110

Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10Bibliographically approved
Nazaryan-Petersen, L., Eisfeldt, J., Lundin, J., Pettersson, M., Nilsson, D., Wincent, J., . . . Lindstrand, A. (2018). Whole genome characterization of array defined clustered CNVs reveals two distinct complex rearrangement subclasses generated through either non-homologous repair or template switching. Paper presented at 50th European-Society-of-Human-Genetics (ESHG) Conference, MAY 27-30, 2017, Copenhagen, Denmark. European Journal of Human Genetics, 26, 60-60
Open this publication in new window or tab >>Whole genome characterization of array defined clustered CNVs reveals two distinct complex rearrangement subclasses generated through either non-homologous repair or template switching
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2018 (English)In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 26, p. 60-60Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-270690 (URN)000489312600117 ()
Conference
50th European-Society-of-Human-Genetics (ESHG) Conference, MAY 27-30, 2017, Copenhagen, Denmark
Note

QC 20200312

Available from: 2020-03-12 Created: 2020-03-12 Last updated: 2020-03-12Bibliographically approved
Eisfeldt, J., Nazaryan-Petersen, L., Lundin, J. L., Pettersson, M., Nilsson, D., Wincent, J., . . . Lindstrand, A. (2017). Whole genome characterization of array defined clustered CNVs reveals two distinct complex rearrangement subclasses generated through either non homologous repair or template switching. Molecular Cytogenetics, 10
Open this publication in new window or tab >>Whole genome characterization of array defined clustered CNVs reveals two distinct complex rearrangement subclasses generated through either non homologous repair or template switching
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2017 (English)In: Molecular Cytogenetics, ISSN 1755-8166, E-ISSN 1755-8166, Vol. 10Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2017
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-215481 (URN)000410864800024 ()
Note

QC 20171013

Available from: 2017-10-13 Created: 2017-10-13 Last updated: 2017-11-29Bibliographically approved
Nilsson, D., Pettersson, M., Gustavsson, P., Förster, A., Hofmeister, W., Wincent, J., . . . Lindstrand, A. (2017). Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation. Human Mutation, 38(2), 180-192
Open this publication in new window or tab >>Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation
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2017 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 38, no 2, p. 180-192Article in journal (Refereed) Published
Abstract [en]

Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low-coverage mate pair whole-genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX-male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error-prone replication-based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keywords
balanced chromosomal aberration, microhomology, nonhomologous end joining, reciprocal translocation, replication-based repair mechanisms, whole-genome sequencing
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-201964 (URN)10.1002/humu.23146 (DOI)000393687800007 ()27862604 (PubMedID)2-s2.0-85006421176 (Scopus ID)
Note

Funding text: Contract grant sponsors: Swedish Research Council [2012-1526, 2013-2603]; The Swedish Society for Medical Research; the Marianne and Marcus Wallenberg Foundation [2014.0084]; Stockholm County Council; the Harald and Greta Jeanssons Foundation; the Ulf Lundahl memory fund through the Swedish Brain Foundation; the Nilsson Ehle donations and the Erik Rönnberg Foundation; US National Institutes of Health [HG006542] to the Baylor Hopkins Center for Mendelian Genomics. We are grateful to the patients and their families for their cooperation and enthusiasm during this study. We also gratefully acknowledge the use of computer infrastructure resources at UPPMAX, projects b2011162 and b2014152.

QC 20170303

Available from: 2017-03-03 Created: 2017-03-03 Last updated: 2020-03-09Bibliographically approved
Hofmeister, W., Nilsson, D., Topa, A., Anderlid, B.-M., Darki, F., Matsson, H., . . . Lindstrand, A. (2015). CTNND2: a candidate gene for reading problems and mild intellectual disability. J MED GENET, 52(2), 111-122
Open this publication in new window or tab >>CTNND2: a candidate gene for reading problems and mild intellectual disability
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2015 (English)In: J MED GENET, ISSN 0022-2593, Vol. 52, no 2, p. 111-122Article in journal (Refereed) Published
Abstract [en]

Background Cytogenetically visible chromosomal translocations are highly informative as they can pinpoint strong effect genes even in complex genetic disorders. Methods and results Here, we report a mother and daughter, both with borderline intelligence and learning problems within the dyslexia spectrum, and two apparently balanced reciprocal translocations: t(1;8)(p22; q24) and t(5; 18)(p15; q11). By low coverage mate-pair whole-genome sequencing, we were able to pinpoint the genomic breakpoints to 2 kb intervals. By direct sequencing, we then located the chromosome 5p breakpoint to intron 9 of CTNND2. An additional case with a 163 kb microdeletion exclusively involving CTNND2 was identified with genome-wide array comparative genomic hybridisation. This microdeletion at 5p15.2 is also present in mosaic state in the patient's mother but absent from the healthy siblings. We then investigated the effect of CTNND2 polymorphisms on normal variability and identified a polymorphism (rs2561622) with significant effect on phonological ability and white matter volume in the left frontal lobe, close to cortical regions previously associated with phonological processing. Finally, given the potential role of CTNND2 in neuron motility, we used morpholino knockdown in zebrafish embryos to assess its effects on neuronal migration in vivo. Analysis of the zebrafish forebrain revealed a subpopulation of neurons misplaced between the diencephalon and telencephalon. Conclusions Taken together, our human genetic and in vivo data suggest that defective migration of subpopulations of neuronal cells due to haploinsufficiency of CTNND2 contribute to the cognitive dysfunction in our patients.

National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-160375 (URN)10.1136/jmedgenet-2014-102757 (DOI)000348203900006 ()25473103 (PubMedID)2-s2.0-84922745112 (Scopus ID)
Funder
Swedish Research Council, 2012-1526 2010-3286 2012-2279 2011-4592
Note

QC 20150227

Available from: 2015-02-27 Created: 2015-02-19 Last updated: 2015-02-27Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-3811-5439

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