| 1. |
- Lindstrand, Anna, et al.
(författare)
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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
- 2019
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Ingår i: Genome Medicine. - : BMC. - 1756-994X .- 1756-994X. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Norling, Ameli
(författare)
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Molecular mechanisms in disorders of gonadal development
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Controlling fertility, or rather infertility, is a common problem for couples nowadays where approximately 10% experience infertility. This thesis focuses on the rare conditions leading to infertility in which the gonads have either failed to develop properly, or prematurely lost their function. The overall aim of this thesis was to identify and study candidate genes in gonadal disorders of sex development (DSD) and primary ovarian insufficiency (POI) to achieve a better understanding of the underlying molecular mechanisms and to improve available diagnostic tools and genetic counseling for affected patients and family members. The process of sex determination is sensitive to gene dosage, and genes affecting gonadal development are located on sex chromosomes and autosomes. Attempting to identify new candidate regions for testicular development, nine patients with 46,XY complete gonadal dysgenesis were investigated with a customized 1M array-CGH (comparative genomic hybridization) platform with whole-genome coverage and probe enrichment targeting 78 genes involved in DSD. Aberrations affecting SUPT3H and C2ORF80 were identified and characterized in two affected sisters. Sequence analysis of these genes in all patients revealed no additional mutations. PIP5K1B and PRKACG were also identified as candidate genes in another patient. All four genes are expressed in testicular tissues, and one is shown to cause gonadal DSD in mice (Paper I). Based on the report of one patient with 46,XY ovarian DSD due to CBX2 mutations and the corresponding knock out mouse model, we considered CBX2 as a candidate gene for 46,XY/46,XX gonadal DSD and POI. By sequencing and MLPA a cohort of 47 patients with gonadal DSD or POI was investigated for sequence alterations and copy number changes in CBX2. No causative mutation was detected. Our study does therefore not support CBX2 mutations as a common cause of gonadal DSD (Paper II). DNA samples from 26 patients with POI were analyzed by our customized array-CGH platform to identify novel candidate genes in POI. Eleven unique copy number changes were identified in a total of 13 patients. We identified the first mutation affecting the regulatory region of GDF9, a 475bp duplication containing three NOBOX binding elements and a regulatory E-box. In addition, we found the second DNAH6 deletion, corroborating its potential role in causing in POI. Also TSPYL6, SMARCC1, CSPG5, SH3GL3 and ZFR2 were identified as candidate genes in POI (Paper III). PSMC3IP mutations have been described causing autosomal recessive POI in a consanguineous family with several affected members. We are the first to have investigated the PSMC3IP gene in a group of unrelated patients with POI. No mutation was detected and we therefore conclude that PSMC3IP mutations are not a common cause of POI in a Swedish patient cohort (Paper IV). In conclusion, there is accumulating evidence that gonadal DSD and POI are highly genetically heterogeneous. We recommend that the genetic investigation of patients with gonadal DSD should be centralized to a specialized DSD unit. Patients with POI should be handled by subspecialists within reproductive endocrinology and clinical genetic investigation can require genetic counseling. A suggested clinical investigation procedure for POI is presented. Ideally, genetic investigation of patients with gonadal DSD or POI should include investigation for gene dosage aberrations as well sequencing of several candidate genes.
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| 3. |
- Norling, Ameli, et al.
(författare)
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Novel candidate genes for 46,XY gonadal dysgenesis identified by a customized 1 M array-CGH platform
- 2013
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Ingår i: European Journal of Medical Genetics. - : Elsevier BV. - 1769-7212 .- 1878-0849. ; 56:12, s. 661-668
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Tidskriftsartikel (refereegranskat)abstract
- Half of all patients with a disorder of sex development (DSD) do not receive a specific molecular diagnosis. Comparative genomic hybridization (CGH) can detect copy number changes causing gene haploinsufficiency or over-expression that can lead to impaired gonadal development and gonadal DSD. The purpose of this study was to identify novel candidate genes for 46,XY gonadal dysgenesis (GD) using a customized 1 M array-CGH platform with whole-genome coverage and probe enrichment targeting 78 genes involved in sex development. Fourteen patients with 46,XY gonadal DSD were enrolled in the study. Nine individuals were analyzed by array CGH. All patients were included in a follow up sequencing study of candidate genes. Three novel candidate regions for 46,XY GD were identified in two patients. An interstitial duplication of the SUPT3H gene and a deletion of C2ORF80 were detected in a pair of affected siblings. Sequence analysis of these genes in all patients revealed no additional mutations. A large duplication highlighting PIP5K1B, PRKACG and FAM189A2 as candidates for 46, XY GD, were also detected. All five genes are expressed in testicular tissues, and one is shown to cause gonadal DSD in mice. However detailed functional information is lacking for these genes.
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