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Ameur, A., Kloosterman, W. P. & Hestand, M. S. (2019). Single-Molecule Sequencing: Towards Clinical Applications. Trends in Biotechnology, 37(1), 72-85
Open this publication in new window or tab >>Single-Molecule Sequencing: Towards Clinical Applications
2019 (English)In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 37, no 1, p. 72-85Article, review/survey (Refereed) Published
Abstract [en]

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmaco-genomics, cancer, and more.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-373914 (URN)10.1016/j.tibtech.2018.07.013 (DOI)000453919500009 ()30115375 (PubMedID)
Available from: 2019-01-17 Created: 2019-01-17 Last updated: 2019-01-17Bibliographically approved
Stattin, E.-L., Johansson, J., Gudmundsson, S., Ameur, A., Lundberg, S., Bondeson, M.-L. & Wilbe, M. (2018). A novel ECEL1 mutation expands the phenotype of distal arthrogryposis multiplex congenita type 5D to include pretibial vertical skin crease. American Journal of Medical Genetics. Part A, 176(6), 1405-1410
Open this publication in new window or tab >>A novel ECEL1 mutation expands the phenotype of distal arthrogryposis multiplex congenita type 5D to include pretibial vertical skin crease
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2018 (English)In: American Journal of Medical Genetics. Part A, ISSN 1552-4825, E-ISSN 1552-4833, Vol. 176, no 6, p. 1405-1410Article in journal (Refereed) Published
Abstract [en]

Arthrogryposis multiplex congenita (AMC) is a heterogeneous disorder characterized by multiple joint contractures often in association with other congenital abnormalities. Pretibial linear vertical creases are a rare finding associated with arthrogryposis, and the etiology of the specific condition is unknown. We aimed to genetically and clinically characterize a boy from a consanguineous family, presenting with AMC and pretibial vertical linear creases on the shins. Whole exome sequencing and variant analysis revealed homozygous novel missense variants of ECEL1 (c.1163T > C, p.Leu388Pro, NM_004826) and MUSK (c.2572C > T, p.Arg858Cys, NM_005592). Both variants are predicted to have deleterious effects on the protein function, with amino acid positions highly conserved among species. The variants segregated in the family, with healthy mother, father, and sister being heterozygous carriers and the index patient being homozygous for both mutations. We report on a unique patient with a novel ECEL1 homozygous mutation, expanding the phenotypic spectrum of Distal AMC Type 5D to include vertical linear skin creases. The homozygous mutation in MUSK is of unknown clinical significance. MUSK mutations have previously shown to cause congenital myasthenic syndrome, a neuromuscular disorder with defects in the neuromuscular junction.

Place, publisher, year, edition, pages
WILEY, 2018
Keywords
arthrogryposis multiplex congenital, congenital myasthenic syndrome, ECEL1, vertical linear skin creases, whole exome sequencing
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-364512 (URN)10.1002/ajmg.a.38691 (DOI)000434040600017 ()29663639 (PubMedID)
Available from: 2018-11-05 Created: 2018-11-05 Last updated: 2018-11-05Bibliographically approved
Ameur, A., Che, H., Martin, M., Bunikis, I., Dahlberg, J., Höijer, I., . . . Gyllensten, U. B. (2018). De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data. Genes, 9(10), Article ID 486.
Open this publication in new window or tab >>De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data
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2018 (English)In: Genes, ISSN 2073-4425, E-ISSN 2073-4425, Vol. 9, no 10, article id 486Article in journal (Refereed) Published
Abstract [en]

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Keywords
de novo assembly, SMRT sequencing, GRCh38, human reference genome, human whole-genome sequencing, population sequencing, Swedish population
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-369762 (URN)10.3390/genes9100486 (DOI)000448656700024 ()30304863 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2014.0272Swedish Research Council
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-17Bibliographically approved
Höijer, I., Tsai, Y.-C., Clark, T. A., Kotturi, P., Dahl, N., Stattin, E., . . . Ameur, A. (2018). Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing. Human Mutation, 39(9), 1262-1272
Open this publication in new window or tab >>Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing
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2018 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 39, no 9, p. 1262-1272Article in journal (Refereed) Published
Abstract [en]

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.

Keywords
amplification-free sequencing, HTT, Huntington disease, No-Amp Targeted sequencing, repeat expansion, SMRT sequencing, somatic mosaicism, targeted enrichment, targeted sequencing
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-364189 (URN)10.1002/humu.23580 (DOI)000443229000010 ()29932473 (PubMedID)
Available from: 2018-11-07 Created: 2018-11-07 Last updated: 2018-11-16Bibliographically approved
Zaghlool, A., Ameur, A., Wu, C., Westholm, J. O., Niazi, A., Manivannan, M., . . . Feuk, L. (2018). Expression profiling and in situ screening of circular RNAs in human tissues. Scientific Reports, 8, Article ID 16953.
Open this publication in new window or tab >>Expression profiling and in situ screening of circular RNAs in human tissues
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 16953Article in journal (Refereed) Published
Abstract [en]

Circular RNAs (circRNAs) were recently discovered as a class of widely expressed noncoding RNA and have been implicated in regulation of gene expression. However, the function of the majority of circRNAs remains unknown. Studies of circRNAs have been hampered by a lack of essential approaches for detection, quantification and visualization. We therefore developed a target-enrichment sequencing method suitable for screening of circRNAs and their linear counterparts in large number of samples. We also applied padlock probes and in situ sequencing to visualize and determine circRNA localization in human brain tissue at subcellular levels. We measured circRNA abundance across different human samples and tissues. Our results highlight the potential of this RNA class to act as a specific diagnostic marker in blood and serum, by detection of circRNAs from genes exclusively expressed in the brain. The powerful and scalable tools we present will enable studies of circRNA function and facilitate screening of circRNA as diagnostic biomarkers.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-371872 (URN)10.1038/s41598-018-35001-6 (DOI)000450280500013 ()30446675 (PubMedID)
Funder
Swedish Research Council, 2012-4530Swedish Research Council, 2017-01861EU, European Research Council, 282330Knut and Alice Wallenberg Foundation
Available from: 2019-01-07 Created: 2019-01-07 Last updated: 2019-01-07Bibliographically approved
Ardui, S., Ameur, A., Vermeesch, J. R. & Hestand, M. S. (2018). Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Research, 46(5), 2159-2168
Open this publication in new window or tab >>Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics
2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 5, p. 2159-2168Article in journal (Refereed) Published
Abstract [en]

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule realtime (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-351426 (URN)10.1093/nar/gky066 (DOI)000427677100008 ()29401301 (PubMedID)
Available from: 2018-06-01 Created: 2018-06-01 Last updated: 2018-06-01Bibliographically approved
Frykholm, C., Klar, J., Tomanovic, T., Ameur, A. & Dahl, N. (2018). Stereocilin gene variants associated with episodic vertigo: expansion of the DFNB16 phenotype. European Journal of Human Genetics, 26(12), 1871-1874
Open this publication in new window or tab >>Stereocilin gene variants associated with episodic vertigo: expansion of the DFNB16 phenotype
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2018 (English)In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 26, no 12, p. 1871-1874Article in journal (Refereed) Published
Abstract [en]

Vestibular disorders comprise a heterogeneous group of diseases with transient or permanent loss of vestibular function. Vestibulopathy is in most cases associated with migraine, Meniere disease, hereditary ataxias, or sensorineural hearing loss. We identified two brothers and their first cousin affected by hearing loss and episodic vertigo. The brothers were homozygous STRC nonsense variant [c.4027 C> T, p.(Q1343*)], whereas their first cousin was compound heterozygous for the STRC nonsense variant and a 97 kb deletion spanning the entire STRC gene. Clinical investigations confirmed pathological vestibular responses in addition to a characteristic DFNB16 hearing loss. The STRC gene encodes Stereocilin in the cochlea and in the vestibular organ where it ensheathes the kinocilium of the otolithic membranes. Stereocilin is associated with the gel overlaying the vestibular kinocilia, suggesting a role for the protein in sensing balance and spatial orientation. Our findings support such a function for Stereocilin in the vestibular organ and expand the phenotype associated with DFNB16.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Otorhinolaryngology
Identifiers
urn:nbn:se:uu:diva-371126 (URN)10.1038/s41431-018-0256-6 (DOI)000450614800019 ()30250054 (PubMedID)
Funder
Swedish Research Council, 2015-02424The Swedish Brain FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-12-20 Created: 2018-12-20 Last updated: 2018-12-20Bibliographically approved
Papoutsoglou, P., Tsubakihara, Y., Caja, L., Pallis, P., Ameur, A., Heldin, C.-H. & Moustakas, A. (2018). The TGFB2-AS1 lncRNA regulates TGFβ signaling by modulating corepressor activity.
Open this publication in new window or tab >>The TGFB2-AS1 lncRNA regulates TGFβ signaling by modulating corepressor activity
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2018 (English)Article in journal (Refereed) Submitted
Abstract [en]

LncRNAs regulate cell function through many physiological processes. We have identified lncRNAs whose expression is regulated by transforming growth factor β (TGFβ), by a transcriptomic screen. We focused on TGFB2-antisense RNA1 (TGFB2-AS1), which was induced by TGFβ through Smad and protein kinase pathways, and exhibited predominantly nuclear localization. Depleting TGFB2-AS1 enhanced TGFβ/Smad-mediated transcription and expression of the TGFβ-target genes FN1 and SERPINE1. Overexpression of TGFB2-AS1 reduced expression of these genes, attenuated TGFβ-induced cell growth arrest, and altered BMP and Wnt pathway gene profiles. Mechanistically, TGFB2-AS1 mainly via its 3’ terminal region, bound to EED, an adaptor of the Polycomb repressor complex 2 (PRC2), promoting repressive histone H3K27me3 modifications at TGFβ-target gene promoters. Silencing EED or inhibiting PRC2 methylation activity, partially rescued TGFB2-AS1 mediated gene repression. Our observations support the notion that TGFB2-AS1 is a TGFβ-induced lncRNA with inhibitory functions on TGFβ and BMP pathways output, constituting an auto-regulatory negative feedback mechanism that balances TGFβ- and BMP-mediated responses.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-363700 (URN)
Available from: 2018-10-20 Created: 2018-10-20 Last updated: 2018-12-03
Bondeson, M.-L., Ericson, K., Gudmundsson, S., Ameur, A., Ponten, F., Wesström, J., . . . Wilbe, M. (2017). A nonsense mutation in CEP55 defines a new locus for a Meckel-like syndrome, an autosomal recessive lethal fetal ciliopathy.. Clinical Genetics, 92(5), 510-516
Open this publication in new window or tab >>A nonsense mutation in CEP55 defines a new locus for a Meckel-like syndrome, an autosomal recessive lethal fetal ciliopathy.
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2017 (English)In: Clinical Genetics, ISSN 0009-9163, E-ISSN 1399-0004, Vol. 92, no 5, p. 510-516Article in journal (Refereed) Published
Abstract [en]

Mutations in genes involved in the cilium-centrosome complex are called ciliopathies. Meckel-Gruber syndrome (MKS) is a ciliopathic lethal autosomal recessive syndrome characterized by genetically and clinically heterogeneous manifestations, including renal cystic dysplasia, occipital encephalocele and polydactyly. Several genes have previously been associated with MKS and MKS-like phenotypes, but there are still genes remaining to be discovered. We have used whole exome sequencing (WES) to uncover the genetics of a suspected autosomal recessive Meckel syndrome phenotype in a family with two affected fetuses. RNA studies and histopathological analysis was performed for further delineation. WES lead to identification of a homozygous nonsense mutation c.256C>T (p.Arg86*) in CEP55 (centrosomal protein of 55 kDa) in the affected fetus. The variant has previously been identified in carriers in low frequencies, and segregated in the family. CEP55 is an important centrosomal protein required for the mid-body formation at cytokinesis. Our results expand the list of centrosomal proteins implicated in human ciliopathies and provide evidence for an essential role of CEP55 during embryogenesis and development of disease.

Keywords
CEP55, Meckel-like, ciliopathy, cytokinesis, whole exome sequencing
National Category
Clinical Medicine Clinical Laboratory Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-318127 (URN)10.1111/cge.13012 (DOI)000412590300007 ()28295209 (PubMedID)
Funder
Magnus Bergvall FoundationLars Hierta Memorial FoundationSwedish Society for Medical Research (SSMF)
Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2019-01-04Bibliographically approved
Wilbe, M., Gudmundsson, S., Johansson, J., Ameur, A., Stattin, E.-L., Annerén, G., . . . Bondeson, M.-L. (2017). A novel approach using long-read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders. Prenatal Diagnosis, 37(11), 1146-1154
Open this publication in new window or tab >>A novel approach using long-read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders
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2017 (English)In: Prenatal Diagnosis, ISSN 0197-3851, E-ISSN 1097-0223, Vol. 37, no 11, p. 1146-1154Article in journal (Refereed) Published
Abstract [en]

Objective

De novo mutations contribute significantly to severe early-onset genetic disorders. Even if the mutation is apparently de novo, there is a recurrence risk due to parental germ line mosaicism, depending on in which gonadal generation the mutation occurred.

Methods

We demonstrate the power of using SMRT sequencing and ddPCR to determine parental origin and allele frequencies of de novo mutations in germ cells in two families whom had undergone assisted reproduction.

Results

In the first family, a TCOF1 variant c.3156C>T was identified in the proband with Treacher Collins syndrome. The variant affects splicing and was determined to be of paternal origin. It was present in <1% of the paternal germ cells, suggesting a very low recurrence risk. In the second family, the couple had undergone several unsuccessful pregnancies where a de novo mutation PTPN11 c.923A>C causing Noonan syndrome was identified. The variant was present in 40% of the paternal germ cells suggesting a high recurrence risk.

Conclusions

Our findings highlight a successful strategy to identify the parental origin of mutations and to investigate the recurrence risk in couples that have undergone assisted reproduction with an unknown donor or in couples with gonadal mosaicism that will undergo preimplantation genetic diagnosis.

National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-342916 (URN)10.1002/pd.5156 (DOI)000415897200012 ()28921562 (PubMedID)
Funder
Swedish Society for Medical Research (SSMF)
Available from: 2018-02-26 Created: 2018-02-26 Last updated: 2018-02-26Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6085-6749

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