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Developmental Biology - DNA Repeats
DNA Repeats Are Equivalent to 'Dark Matter' In Space
A new analysis of DNA sequence repeats tells us how they cause disease in humans...
DNA repeats in our genome are very hard to analyze as they can expand to hugh lengths. But now, a new method of analysis has been developed by researchers at the Max Planck Institute for Molecular Genetics in Berlin, Germany. It is giving us a detailed look at these previously inaccessible DNA repeats on our genome.
This new method combines nanopore sequencing, stem cell and CRISPR-Cas technologies. In the future it could improve the diagnosis of various congenital diseases and cancers.
Large parts of our genome consist of monotonous regions where short sections of it repeat hundreds or thousands of times.
But expansions of these "DNA repeats" in the wrong places can have dramatic consequences. Such as in those people with Fragile X syndrome, one of the most commonly identifiable hereditary causes of cognitive disability in humans.
However, repetitive regions are still regarded as unknown territory. They can't be easily examined — even with modern methods.
So a research team led by Franz-Josef Müller PhD at the Max Planck Institute for Molecular Genetics in Berlin, along with the University Hospital of Schleswig-Holstein in Kiel, recently shed new light on these inaccessible genomic regions.
Müller's team was the first to successfully determine the length of theses genomic repeats. Using patient-derived stem cells grown in culture, his researchers scanned individual DNA molecules using nanopore sequencing and CRISPR-Cas technology to cut apart the gene. His method opens research into these repetitive gene regions, leading research in a fast and accurate diagnosis of a range of diseases.
A Gene Defect On The X Chromosome
Fragile X syndrome is the result of an expanded DNA sequence on the FMR1 gene located on the X chromosome.
"The cell recognizes this repetitive region and switches it off by attachment of methyl groups to the DNA. Unfortunately, the epigenetic marks spread over the entire gene, which then completely shuts down. Without the FMR1 gene, we see severe delays in development leading to varying degrees of intellectual disability or autism."
Franz-Josef Müller PhD,
Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany.
The FMR1 gene is essential for normal brain development, states Müller.
Females are, in most cases, less affected by Fragile X as the repeat region is usually located on only one of the two X chromosomes. Since the unchanged second copy of the gene is not altered, it compensates for the genetic defect on the other X chromosome.
Males having only one X chromosome, and so only the copy of the affected gene, display the full range of clinical symptoms. Fragile X syndrome is one of about 30 diseases caused by expanding short DNA tandem repeats.
First Precise Map of Short Tandem Repeats
In the study, appearing in Nature Biotechnology, Müller and his team investigated the genome of stem cells derived from patient tissue. With this tissue they were able to determine (1) length of repeat regions and (2) their epigenetic signature — a feat not possible with conventional sequencing methods. Researchers also discovered the length of the repetitive region could vary to a large degree, even among cells from a single patient.
Researchers also tested their technology on cells derived from patients with frontotemporal dementia and amyotrophic lateral sclerosis. Each contained expanded DNA repeats in one of the two copies of the C9orf72 gene, with a mutation leading to one of the most common monogenic causes of either disorder.
Tiny Pores Scan Single Molecules
"Conventional methods are limited when it comes to highly repetitive DNA sequences."
Björn Brändl
Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany; and one of the first authors of the publication.
That's why the scientists used Nanopore sequencing technology capable of analyzing these regions. The DNA is fragmented, and each strand is threaded through one of a hundred tiny holes ("nanopores") on a silicon chip. At the same time, electrically charged particles flow through the pores and generate a current.
When a DNA molecule moves through one of these pores, that current varies depending on the chemical properties of the DNA. Fluctuations in the electrical signal are enough for the computer to reconstruct a genetic sequence and epigenetic chemical label. This process takes place at each pore, identifying each strand of DNA.
Genome Editing Tools Illuminate "Dark Matter"
Conventional sequencing methods already can analyze the entire genome of a patient. But now, scientists have designed a process to look at specific regions selectively.
Brändl used the CRISPR-Cas system to cut DNA segments from the genome that contained the repeat region. These segments went through a few intermediate processing steps and were then funneled into the pores on the sequencing chip.
"If we had not pre-sorted molecules in this way, their signal would have been drowned by the noise of the rest of the genome. Most algorithms fail because they do not expect the regular patterns of repetitive sequences."
Pay Giesselmann PhD, Bioinformatician, Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany.
Giesselmann's program "STRique" does not determine the genetic sequence itself, but counts the number of sequence repetitions with high precision. The program is freely available on the internet.
Numerous Applications in Research at the Clinic
"With the CRISPR-Cas system and our algorithms, we can scrutinize any section of the genome - especially those regions that are particularly difficult to examine using conventional methods.
We created tools that enable every researcher to explore the dark matter in the genome. There is evidence that the repeats grow during the development of the nervous system, and we would like to take a closer look at this."
Franz-Josef Müller PhD,
Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany, and head of the project.
As repetitive regions are involved in the development of cancer, and the new method is relatively inexpensive and fast, Müller is determined to take the procedure to the next level: "We are very close to clinical application."
Abstract
Expansions of short tandem repeats are genetic variants that have been implicated in several neuropsychiatric and other disorders, but their assessment remains challenging with current polymerase-based methods1,2,3,4. Here we introduce a CRISPR–Cas-based enrichment strategy for nanopore sequencing combined with an algorithm for raw signal analysis. Our method, termed STRique for short tandem repeat identification, quantification and evaluation, integrates conventional sequence mapping of nanopore reads with raw signal alignment for the localization of repeat boundaries and a hidden Markov model-based repeat counting mechanism. We demonstrate the precise quantification of repeat numbers in conjunction with the determination of CpG methylation states in the repeat expansion and in adjacent regions at the single-molecule level without amplification. Our method enables the study of previously inaccessible genomic regions and their epigenetic marks.
Authors
Pay Giesselmann, Björn Brändl, Etienne Raimondeau, Rebecca Bowen, Christian Rohrandt, Rashmi Tandon, Helene Kretzmer, Günter Assum, Christina Galonska, Reiner Siebert, Ole Ammerpohl, Andrew Heron, Susanne A. Schneider, Julia Ladewig, Philipp Koch, Bernhard M. Schuldt, James E. Graham, Alexander Meissner and Franz-Josef Müller.
Acknowledgments
The authors deeply thank the invaluable support by the patients with c9FTD/ALS and FXS and their families who donated biomaterials for this study. The C9orf72 BAC was generously provided by R. Baloh and S. Bell (Cedars Sinai Medical Center, Los Angeles, CA, USA). We are grateful to J. Loring and A. Zhang (Scripps Research Institute, La Jolla, CA, USA) for providing us with hiPSC lines from a patient with FXS (supported by NIH R33MH087925-03). We thank P. van Damme and W. Robberecht (Laboratory for Neurobiology; VIB-KU Leuven Center for Brain & Disease Research, Belgium) for providing the fibroblasts derived from patients with c9FTD/ALS used for reprogramming. We acknowledge the expert assistance of the technical staff of the Molecular Genetics Laboratory of the Institute of Human Genetics (Ulm, Germany). P.K. and J.L. acknowledge financial support by the Hector Stiftung II gGmbH. F.J.M. and R.T. received funding from the Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy–EXC 22167-390884018. F.J.M. and B.M.S. were supported by the BMBF (PluriTest2, 13GW0128A). This work was supported by the Max Planck Society. This work was overseen and approved by the Ethics Committee of the Christian-Albrechts-University (Kiel, Germany; reference no. A 145/11). Informed consent was obtained from all donors of cells and tissues used for the generation of hiPSC lines and their subsequent genetic and epigenetic analysis. All materials were donated graciously by our patients.
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