Clinical Trial Details
— Status: Enrolling by invitation
Administrative data
NCT number |
NCT06072079 |
Other study ID # |
2019-02078 |
Secondary ID |
|
Status |
Enrolling by invitation |
Phase |
|
First received |
|
Last updated |
|
Start date |
December 20, 2019 |
Est. completion date |
December 2029 |
Study information
Verified date |
October 2023 |
Source |
Karolinska Institutet |
Contact |
n/a |
Is FDA regulated |
No |
Health authority |
|
Study type |
Observational
|
Clinical Trial Summary
The project is focused on the detailed study of structural genomic variants (SVs). Such
genetic mutations are in fact alterations in the DNA molecule structure and include copy
number variants, inversions and translocations. A single event may affect many genes as well
as regulatory regions and the specific phenotypic consequences will depend on the location,
genetic content and type of SV. Many times, the specific disease-causing mechanism is not
known. Here, we plan to study the molecular genetic behavior of structural variants as well
as the underlying mutational mechanisms involved.
First, we will use genome sequencing to pinpoint the chromosomal breakpoints at the
nucleotide level, characterize the genomic architecture at the breakpoints and study the
relationship between structural variants and SNVs. Second, we will study how structural
variants impact gene expression. Finally, we will functionally explore the disease mechanisms
in vivo using zebrafish and in vitro using primary patient cells and induced pluripotent stem
cells.
Our studies will focus on the origin, structure and impact of structural variation on human
disease. The results will directly lead to a higher mutation detection rate in genetic
diagnostics. Through a better understanding of disease mechanisms our findings will also
assist in the development of novel biomarkers and therapeutic strategies for patients with
rare genetic disorders.
Description:
PROJECT DESCRIPTION Aim 1) What are the molecular mechanisms of formation for structural
genomic variants?
We already have WGS data from more than 500 individual SVs. To understand underlying
mutational and disease mechanisms, we will combine structural variant detection with
positional information for the rearrangement. This will be particularly important for
duplications (the location of the duplicated genomic segment is key to a correct clinical
interpretation) and for complex rearrangements. Second, the exact breakpoint will be
established and breakpoint junctions analyzed for sequence motifs.
Aim 2) What are the molecular mechanisms involved in disease pathology?
First we will use WGS to identify and characterize structural variants. Next, to understand
how the candidate disease gene(s) affected by the SVs cause clinical symptoms, we will study
the cellular mechanisms involved in disease pathology in three ways:
Part 1. In vitro studies of primary cells: Effects on expression and splicing, will be
evaluated in patient cells (fibroblasts or B cell lines) with RT-PCR, qPCR and Western blot.
Part 2. In vitro studies in patient-derived models: In selected cases, we will generate
induced-pluripotent stem cells and neural progenitor cells (NES cells), which will provide an
opportunity to directly study the effects of early neurogenesis in cells from the patient and
compare to cells derived from normal controls. We are interested in studying both early
neurogenesis in 2D culture using the NES cells, as well as generating another developmentally
relevant model, brain organoids (mini brains), in 3D culture directly from iPS cell lines.
Organoid 3D culture recapitulates development of various brain regions and is therefore a
unique tool to investigate brain disorders. Furthermore, the 3D neural culture could improve
cell maturity and stimulate expression of disease phenotypes that facilitate better
understanding the disease mechanisms.
Part 3. In vivo studies in a zebrafish model: Zebrafish (Danio rerio) is a well-functioning
in vivo system for studies of normal and abnormal embryological development. Duplications are
simulated by RNA overexpression and deletion through CRISPR/Cas9 induced gene disruption or
morpholino knock down. Assessment of the phenotypes is designed in accordance with the those
observed in patients (e.g. head size, craniofacial defects, cardiovascular malformations,
cilia defects).
Aim 3) How does structural genomic variants impact gene expression?
We plan to study long range effects in three ways. Part 1. To assess the clinical impact of
TAD disruptions we will search for such events in the CNV data from the Clinical Genetics
array database (data from over 6200 children with rare NDDs), LocusDB SV (SV calls from >1000
patients with rare diseases analyzed by WGS at Clinical Genomics SciLifeLab) and publicly
available databases (DECIPHER, ICCG and SWEGEN). The Bioinformatic analysis will combine
information on phenotypic overlap between known-disease genes surrounding the SV and the
patients' characteristics, as well as on physical overlap with tissue-specific enhancers and
TAD regions. Novel computational approaches will be designed for the discovery of genes
disrupted by similar mechanisms. Follow-up studies will involve further patient
characterization and customized RNA studies.
Part 2. Transcriptome analysis of patient samples is done by RNA-seq. Since not every gene is
transcriptionally active in every cell, a key to successfully achieving this aim is access to
biologically relevant samples. Often biopsies are not available therefore, in those cases,
iPS cells are an alternative way to study biologically relevant effects on RNA expression. In
cases with paired WGS and RNA-seq data, we will integrate changes in transcription levels and
ASE as a readout for the SVs effect on neighboring genes.
Part 3. Cell and animal studies: Introduction of specific SVs with CRISPR/Cas9 in human cell
lines will be done when the primary cells are unavailable and zebrafish lines created under
goal 2 may also be used to evaluate global effects of SVs on an organism level.