Complex chromosomal rearrangements (CCRs) are rearrangements involving more than two chromosomes or more than two breakpoints. Whole genome sequencing (WGS) allows for outstanding high resolution ...characterization on the nucleotide level in unique sequences of such rearrangements, but problems remain for mapping breakpoints in repetitive regions of the genome, which are known to be prone to rearrangements. Hence, multiple complementary WGS experiments are sometimes needed to solve the structures of CCRs. We have studied three individuals with CCRs: Case 1 and Case 2 presented with de novo karyotypically balanced, complex interchromosomal rearrangements (46,XX,t(2;8;15)(q35;q24.1;q22) and 46,XY,t(1;10;5)(q32;p12;q31)), and Case 3 presented with a de novo, extremely complex intrachromosomal rearrangement on chromosome 1. Molecular cytogenetic investigation revealed cryptic deletions in the breakpoints of chromosome 2 and 8 in Case 1, and on chromosome 10 in Case 2, explaining their clinical symptoms. In Case 3, 26 breakpoints were identified using WGS, disrupting five known disease genes. All rearrangements were subsequently analyzed using optical maps, linked-read WGS, and short-read WGS. In conclusion, we present a case series of three unique de novo CCRs where we by combining the results from the different technologies fully solved the structure of each rearrangement. The power in combining short-read WGS with long-molecule sequencing or optical mapping in these unique de novo CCRs in a clinical setting is demonstrated.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The clinical utilization of non-invasive prenatal testing (NIPT) for identification of fetal aneuploidies is expanding worldwide. The aim of this study was to gain an increased understanding of ...pregnant women's awareness, attitudes, preferences for risk information and decision-making concerning prenatal examinations with emphasis on NIPT, before its introduction into Swedish healthcare.
Pregnant women were recruited to fill in a questionnaire, including multiple-choice questions and Likert scales, at nine maternity clinics located in different areas of Stockholm, Sweden.
In total, 1,003 women participated in the study (86% consent rate). The vast majority (90.7%) considered examinations aiming to detect fetal abnormalities to be good. Regarding NIPT, 59.8% stated that they had heard about the method previously, yet 74.0% would like to use the test if available. The main factor affecting the women's decision to undergo prenatal chromosomal screening was worry about the baby's health (82.5%), followed by the urge to have as much information as possible about the fetus (54.5%). Most women (79.9%) preferred to receive NIPT information orally.
The overwhelming majority of a cohort of 1,003 pregnant women considered prenatal examinations good. Moreover, the majority had a positive attitude towards NIPT and would like to use the test if available.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
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.
We use whole genome sequencing to characterize reciprocal translocations in both healthy and affected carriers. First, we show that half the unaffected carriers harbored disruptions in recessive disease genes and in 20% of the affected individuals the phenotype was explained by disruption of a dominant disease gene. In addition, seven candidate disease genes are pinpointed by a chromosome breakpoint. Second, we analyze the breakpoint junction sequences and identify a ‘mutational signature’ consistent with an underlying mechanism involving template switching.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CASK-related disorders are genetically defined neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate ...CASK-mutation consequences and neuronal effects using induced pluripotent stem cell-derived neurons from two mutation carriers. One male case with autism spectrum disorder carried a novel splice-site mutation and a female case with intellectual disability carried an intragenic tandem duplication. We show reduction of CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of genes involved in presynaptic development and of CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory-inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in the brain. Our data show that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.
The pyruvate dehydrogenase complex (PDC) is responsible for the conversion of pyruvate into acetyl-CoA, which is used for energy conversion in cells. PDC activity is regulated by phosphorylation via ...kinases and phosphatases (PDK/PDP). Variants in all subunits of the PDC and in PDK3 have been reported, with varying phenotypes including lactic acidosis, neurodevelopmental delay, peripheral neuropathy, or seizures. Here, we report a de novo heterozygous missense variant in PDK1 (c.1139G > A; p.G380D) in a girl with developmental delay and early onset severe epilepsy. To investigate the role of PDK1G380D in energy metabolism and neuronal development, we used a zebrafish model. In zebrafish embryos we show a reduced number of cells with mitochondria with membrane potential, reduced movements, and a delay in neuronal development. Furthermore, we observe a reduction in the phosphorylation of PDH-E1α by PDKG380D, which suggests a disruption in the regulation of PDC activity. Finally, in patient fibroblasts, a mild reduction in the ratio of phosphorylated PDH over total PDH-E1α was detected. In summary, our findings support the notion that this aberrant PDK1 activity is the cause of clinical symptoms in the patient.
DNA double strand breaks (DSBs) are mainly repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ). These breaks pose severe threats to genome integrity but can also be ...necessary intermediates of normal cellular processes such as immunoglobulin class switch recombination (CSR). During CSR, DSBs are produced in the G1 phase of the cell cycle and are repaired by the classical NHEJ machinery. By studying B lymphocytes derived from patients with Cornelia de Lange Syndrome, we observed a strong correlation between heterozygous loss-of-function mutations in the gene encoding the cohesin loading protein NIPBL and a shift toward the use of an alternative, microhomology-based end joining during CSR. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Association of NIPBL deficiency and impaired NHEJ was also observed in a plasmid-based end-joining assay and a yeast model system. Our results suggest that NIPBL plays an important and evolutionarily conserved role in NHEJ, in addition to its canonical function in sister chromatid cohesion and its recently suggested function in HR.
We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases ...(GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting.
Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams.
Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange.
Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.
Cornelia de Lange syndrome (CdLS; OMIM 122470) is a rare multiple congenital anomaly/mental retardation syndrome characterized by distinctive dysmorphic facial features, severe growth and ...developmental delay and abnormalities of the upper limbs. About 50% of CdLS patients have been found to have heterozygous mutations in the NIPBL gene and a few cases were recently found to be caused by mutations in the X-linked SMC1L1 gene. We performed a mutation screening of all NIPBL coding exons by direct sequencing in 11 patients (nine sporadic and two familial cases) diagnosed with CdLS in Sweden and detected mutations in seven of the cases. All were de novo, and six of the mutations have not been previously described. Four patients without identifiable NIPBL mutations were subsequently subjected to multiplex ligation-dependent probe amplification analysis to exclude whole exon deletions/duplications of NIPBL. In addition, mutation analysis of the 5' untranslated region (5' UTR) of NIPBL was performed. Tiling resolution array comparative genomic hybridization analysis was carried out on these four patients to detect cryptic chromosome imbalances and in addition the boys were screened for SMC1L1 mutations. We found a de novo 9p duplication with a size of 0.6 Mb in one of the patients with a CdLS-like phenotype but no mutations were detected in SMC1L1. So far, two genes (NIPBL and SMC1L1) have been identified causing CdLS or CdLS-like phenotypes. However, in a considerable proportion of individuals demonstrating the CdLS phenotype, mutations in any of these two genes are not found and other potential loci harboring additional CdLS-causing genes should be considered.
Since 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.
We 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.
First, 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 850 kb (min 500 bp, max 155 Mb). 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 (> 10 kb) 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.
The 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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