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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
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
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: 2018-01-08Bibliographically 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
Pan, G., Ameur, A., Enroth, S., Bysani, M., Nord, H., Cavalli, M., . . . Wadelius, C. (2017). PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c. Nucleic Acids Research, 45(5), 2408-2422
Open this publication in new window or tab >>PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c
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2017 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 5, p. 2408-2422Article in journal (Refereed) Published
Abstract [en]

The FADS1 and FADS2 genes in the FADS cluster encode the rate-limiting enzymes in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). Genetic variation in this region has been associated with a large number of diseases and traits many of them correlated to differences in metabolism of PUFAs. However, the causative variants leading to these associations have not been identified. Here we find that the multiallelic rs174557 located in an AluYe5 element in intron 1 of FADS1 is functional and lies within a PATZ1 binding site. The derived allele of rs174557, which is the common variant in most populations, diminishes binding of PATZ1, a transcription factor conferring allele-specific downregulation of FADS1 The PATZ1 binding site overlaps with a SP1 site. The competitive binding between the suppressive PATZ1 and the activating complex of SP1 and SREBP1c determines the enhancer activity of this region, which regulates expression of FADS1.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-317999 (URN)10.1093/nar/gkw1186 (DOI)000397286600024 ()27932482 (PubMedID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council, 521-2010-3505 6212011-6052 521-2012-2884Swedish Diabetes AssociationSwedish Cancer Society, 15 0878
Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2017-04-18Bibliographically approved
Gudmundsson, S., Wilbe, M., Ekvall, S., Ameur, A., Cahill, N., Alexandrov, L. B., . . . Bondeson, M.-L. (2017). Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26. Human Molecular Genetics, 26(6), 1070-1077
Open this publication in new window or tab >>Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26
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2017 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 26, no 6, p. 1070-1077Article in journal (Refereed) Published
Abstract [en]

Revertant mosaicism(RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c. 148G>A, p. Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultradeep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p. Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p. Asp50Asn mutation. To our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2017
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-324349 (URN)10.1093/hmg/ddx017 (DOI)000400911000004 ()28158657 (PubMedID)
Funder
Swedish Research Council, K2013-57X-22309-3
Available from: 2017-06-15 Created: 2017-06-15 Last updated: 2017-06-15Bibliographically approved
Stattin, E., Henning, P., Klar, J., McDermott, E., Stecksen-Blicks, C., Sandström, P.-E., . . . Lerner, U. H. (2017). SNX10 gene mutation leading to osteopetrosis with dysfunctional osteoclasts.. Scientific Reports, 7, Article ID 3012.
Open this publication in new window or tab >>SNX10 gene mutation leading to osteopetrosis with dysfunctional osteoclasts.
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 3012Article in journal (Refereed) Published
Abstract [en]

Autosomal recessive osteopetrosis (ARO) is a heterogeneous disorder, characterized by defective osteoclastic resorption of bone that results in increased bone density. We have studied nine individuals with an intermediate form of ARO, from the county of Västerbotten in Northern Sweden. All afflicted individuals had an onset in early infancy with optic atrophy, and in four patients anemia was present at diagnosis. Tonsillar herniation, foramen magnum stenosis, and severe osteomyelitis of the jaw were common clinical features. Whole exome sequencing, verified by Sanger sequencing, identified a splice site mutation c.212 + 1 G > T in the SNX10 gene encoding sorting nexin 10. Sequence analysis of the SNX10 transcript in patients revealed activation of a cryptic splice site in intron 4 resulting in a frame shift and a premature stop (p.S66Nfs * 15). Haplotype analysis showed that all cases originated from a single mutational event, and the age of the mutation was estimated to be approximately 950 years. Functional analysis of osteoclast progenitors isolated from peripheral blood of patients revealed that stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) resulted in a robust formation of large, multinucleated osteoclasts which generated sealing zones; however these osteoclasts exhibited defective ruffled borders and were unable to resorb bone in vitro.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-325569 (URN)10.1038/s41598-017-02533-2 (DOI)000402879800068 ()28592808 (PubMedID)
Funder
Swedish Research CouncilSwedish Rheumatism Association
Available from: 2017-06-26 Created: 2017-06-26 Last updated: 2017-09-06Bibliographically approved
Ameur, A., Dahlberg, J., Olason, P., Vezzi, F., Karlsson, R., Martin, M., . . . Gyllensten, U. B. (2017). SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population. European Journal of Human Genetics, 25(11), 1253-1260
Open this publication in new window or tab >>SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population
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2017 (English)In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 25, no 11, p. 1253-1260Article in journal (Refereed) Published
Abstract [en]

Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-337314 (URN)10.1038/ejhg.2017.130 (DOI)000412823800012 ()28832569 (PubMedID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation, 2014.0272Swedish Research CouncilSwedish National Infrastructure for Computing (SNIC), sens2016003EU, European Research Council, 282330
Available from: 2018-01-08 Created: 2018-01-08 Last updated: 2018-07-06Bibliographically approved
Bergfors, A., Leenheer, D., Bergqvist, A., Ameur, A. & Lennerstrand, J. (2016). Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing. Antiviral Research, 126, 81-89
Open this publication in new window or tab >>Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing
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2016 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 126, p. 81-89Article in journal (Refereed) Published
Abstract [en]

Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naive-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naive patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual.

Keywords
Hepatitis C virus, NS5A, Resistance, RAV, Deep sequencing, Pacific biosciences
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-279573 (URN)10.1016/j.antiviral.2015.12.005 (DOI)000369680000010 ()26707078 (PubMedID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2016-03-02 Created: 2016-03-02 Last updated: 2017-11-30Bibliographically approved
Lopes, F., Barbosa, M., Ameur, A., Soares, G., de Sa, J., Dias, A. I., . . . Maciel, P. (2016). Identification of novel genetic causes of Rett syndrome-like phenotypes. Journal of Medical Genetics, 53(3), 190-199
Open this publication in new window or tab >>Identification of novel genetic causes of Rett syndrome-like phenotypes
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2016 (English)In: Journal of Medical Genetics, ISSN 0022-2593, E-ISSN 1468-6244, Vol. 53, no 3, p. 190-199Article in journal (Refereed) Published
Abstract [en]

Background The aim of this work was to identify new genetic causes of Rett-like phenotypes using array comparative genomic hybridisation and a whole exome sequencing approach. Methods and results We studied a cohort of 19 Portuguese patients (16 girls, 3 boys) with a clinical presentation significantly overlapping Rett syndrome (RTT). Genetic analysis included filtering of the single nucleotide variants and indels with preference for de novo, homozygous/compound heterozygous, or maternally inherited X linked variants. Examination by MRI and muscle biopsies was also performed. Pathogenic genomic imbalances were found in two patients (10.5%): an 18q21.2 deletion encompassing four exons of the TCF4 gene and a mosaic UPD of chromosome 3. Variants in genes previously implicated in neurodevelopmental disorders (NDD) were identified in six patients (32%): de novo variants in EEF1A2, STXBP1 and ZNF238 were found in three patients, maternally inherited X linked variants in SLC35A2, ZFX and SHROOM4 were detected in two male patients and one homozygous variant in EIF2B2 was detected in one patient. Variants were also detected in five novel NDD candidate genes (26%): we identified de novo variants in the RHOBTB2, SMARCA1 and GABBR2 genes; a homozygous variant in EIF4G1; compound heterozygous variant in HTT. Conclusions Network analysis reveals that these genes interact by means of protein interactions with each other and with the known RTT genes. These findings expand the phenotypical spectrum of previously known NDD genes to encompass RTT-like clinical presentations and identify new candidate genes for RTT-like phenotypes.

Keywords
Rett syndrome, Epilepsy, Whole exome sequencing, Intellectual disability
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-282471 (URN)10.1136/jmedgenet-2015-103568 (DOI)000371329600006 ()26740508 (PubMedID)
Available from: 2016-04-05 Created: 2016-04-05 Last updated: 2018-01-10Bibliographically approved
Rafati, N., Andersson, L. S., Mikko, S., Feng, C., Raudsepp, T., Pettersson, J., . . . Rubin, C.-J. (2016). Large Deletions at the SHOX Locus in the Pseudoautosomal Region Are Associated with Skeletal Atavism in Shetland Ponies. G3: Genes, Genomes, Genetics, 6(7), 2213-2223
Open this publication in new window or tab >>Large Deletions at the SHOX Locus in the Pseudoautosomal Region Are Associated with Skeletal Atavism in Shetland Ponies
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2016 (English)In: G3: Genes, Genomes, Genetics, ISSN 2160-1836, E-ISSN 2160-1836, Vol. 6, no 7, p. 2213-2223Article in journal (Refereed) Published
Abstract [en]

Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure and impaired movements, and affected foals are usually killed. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds. Read depth analysis at these loci revealed homozygosity or compound heterozygosity for two partially overlapping large deletions in the pseudoautosomal region (PAR) of chromosome X/Y in cases but not in the control pool. One of these deletions removes the entire coding region of the SHOX gene and both deletions remove parts of the CRLF2 gene located downstream of SHOX. The horse reference assembly of the PAR is highly fragmented, and in order to characterize this region we sequenced bacterial artificial chromosome (BAC) clones by single-molecule real-time (SMRT) sequencing technology. This considerably improved the assembly and enabled size estimations of the two deletions to 1602180 kb and 60280 kb, respectively. Complete association between the presence of these deletions and disease status was verified in eight other affected horses. The result of the present study is consistent with previous studies in humans showing crucial importance of SHOX for normal skeletal development.

Keywords
SMRT sequencing, skeletal atavism, SHOX, PAR
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-300547 (URN)10.1534/g3.116.029645 (DOI)000379590200041 ()27207956 (PubMedID)
Available from: 2016-08-10 Created: 2016-08-09 Last updated: 2017-11-28Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6085-6749

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