Molecular cytogenetics

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Image: example of karyotyping showing a total of 46 chromosomes in the genome. DAPIstainedkarotypeofAprasiaparapulchellafemale.jpg
Image: example of karyotyping showing a total of 46 chromosomes in the genome.

Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. [1] [2] It includes a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980s, FISH uses probes with complementary base sequences to locate the presence or absence of the specific DNA regions. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico .

Contents

Common techniques

Fluorescence in situ hybridization (FISH)

FISH images of chromosomes from dividing orangutan (left) and human (right) cells. Yellow probe shows 4 copies of a region in the orangutan genome and only 2 copies in human. Chr2 orang human.jpg
FISH images of chromosomes from dividing orangutan (left) and human (right) cells. Yellow probe shows 4 copies of a region in the orangutan genome and only 2 copies in human.

Fluorescence In Situ Hybridization maps out single copy or repetitive DNA sequences through localization labeling of specific nucleic acids. The technique utilizes different DNA probes labeled with fluorescent tags that bind to one or more specific regions of the genome. [3] It labels all individual chromosomes at every stage of cell division to display structural and numerical abnormalities that may arise throughout the cycle. This is done with a probe that can be locus specific, centromeric, telomeric, and whole-chromosomal. This technique is typically performed on interphase cells and paraffin block tissues. FISH maps out single copy or repetitive DNA sequences through localization labeling of specific nucleic acids. The technique utilizes different DNA probes labeled with fluorescent tags that bind to one or more specific regions of the genome. Signals from the fluorescent tags can be seen with microscopy, and mutations can be seen by comparing these signals to healthy cells. For this to work, DNA must be denatured using heat or chemicals to break the hydrogen bonds; this allows hybridization to occur once two samples are mixed. The fluorescent probes create new hydrogen bonds, thus repairing DNA with their complementary bases, which can be detected through microscopy. FISH allows one to visualize different parts of the chromosome at different stages of the cell cycle. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from microarrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico . [4]

Comparative genomic hybridization (CGH)

Comparative genomic hybridization (CGH), derived from FISH, is used to compare variations in copy number between a biological sample and a reference. CGH was originally developed to observe chromosomal aberrations in tumour cells. This method uses two genomes, a sample and a control, which are labeled fluorescently to distinguish them. [5] In CGH, DNA is isolated from a tumour sample and biotin is attached. Another labelling protein, digoxigenin, is attached to the reference DNA sample. [6] The labelled DNA samples are co-hybridized to probes during cell division, which is the most informative time for observing copy number variation. [7] CGH uses creates a map that shows the relative abundance of DNA and chromosome number. By comparing the fluorescence in a sample compared to a reference, CGH can point to gains or losses of chromosomal regions. [6] [8] CGH differs from FISH because it does not require a specific target or previous knowledge of the genetic region being analyzed. CGH can also scan an entire genome relatively quickly for various chromosome imbalances, and this is helpful in patients with underlying genetic issues and when an official diagnosis is not known. This often occurs with hematological cancers.

Array comparative genomic hybridization (aCGH)

Array comparative genomic hybridization (aCGH) allows CGH to be performed without cell culture and isolation. Instead, it is performed on glass slides containing small DNA fragments. [9] Removing the cell culture and isolation step dramatically simplifies and expedites the process. Using similar principles to CGH, the sample DNA is isolated and fluorescently labelled, then co-hybridized to single stranded probes to generate signals. Thousands of these signals can be detected for at once, and this process is referred to as parallel screening. [10] Fluorescence ratios between the sample and reference signals are measured, representing the average difference between the amount of each. This will show if there is more or less sample DNA than is expected by reference.

Applications

A cell containing a rearrangement of the bcr/abl chromosomal regions (upper left red and green chromosome). This rearrangement is associated with chronic myelogenous leukemia, and was detected using FISH. Bcrablmet.jpg
A cell containing a rearrangement of the bcr/abl chromosomal regions (upper left red and green chromosome). This rearrangement is associated with chronic myelogenous leukemia, and was detected using FISH.

FISH chromosome in-situ hybridization allows the study cytogenetics in pre- and postnatal samples and is also widely used in cytogenetic testing for cancer. While cytogenetics is the study of chromosomes and their structure, cytogenetic testing involves the analysis of cells in the blood, tissue, bone marrow, or fluid to identify changes in chromosomes of an individual. This was often done through karyotyping, and is now done with FISH. This method is commonly used to detect chromosomal deletions or translocations often associated with cancer. FISH is also used for melanocytic lesions, distinguishing atypical melanocytic or malignant melanoma. [5]

Cancer cells often accumulate complex chromosomal structural changes such as loss, duplication, inversion or movement of a segment. [11] When using FISH, any changes to a chromosome will be made visible through discrepancies between fluorescent-labelled cancer chromosomes and healthy chromosomes. [11] The findings of these cytogenetic experiments can shed light on the genetic causes for the cancer and can locate potential therapeutic targets. [12]

Molecular cytogenetics can also be used as a diagnostic tool for congenital syndromes in which the underlying genetic causes of the disease are unknown. [13] Analysis of a patient's chromosome structure can reveal causative changes. New molecular biology methods developed in the past two decades such as next generation sequencing and RNA-seq have largely replaced molecular cytogenetics in diagnostics, but recently the use of derivatives of FISH such as multicolour FISH and multicolour banding (mBAND) has been growing in medical applications. [14]

Cancer projects

One of the current projects involving Molecular Cytogenetics involves genomic research on rare cancers, called the Cancer Genome Characterization Initiative (CGCI). [15] The CGCI is a group interested in describing the genetic abnormalities of some rare cancers, by employing advanced sequencing of genomes, exomes, and transcriptomes, which may ultimately play a role in cancer pathogenesis. [15] Currently, the CGCI has elucidated some previously undetermined genetic alterations in medulloblastoma and B-cell non-Hodgkin lymphoma. The next steps for the CGCI is to identify genomic alternations in HIV+ tumors and in Burkitt's Lymphoma.

Some high-throughput sequencing techniques that are used by the CGCI include: whole genome sequencing, transcriptome sequencing, ChIP-sequencing, and Illumina Infinum MethylationEPIC BeadCHIP. [16]

Related Research Articles

<span class="mw-page-title-main">Cytogenetics</span> Branch of genetics

Cytogenetics is essentially a branch of genetics, but is also a part of cell biology/cytology, that is concerned with how the chromosomes relate to cell behaviour, particularly to their behaviour during mitosis and meiosis. Techniques used include karyotyping, analysis of G-banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH).

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.

Fluorescence <i>in situ</i> hybridization Genetic testing technique

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

<span class="mw-page-title-main">Polysomy</span> Abnormal multiples of one or more chromosomes

Polysomy is a condition found in many species, including fungi, plants, insects, and mammals, in which an organism has at least one more chromosome than normal, i.e., there may be three or more copies of the chromosome rather than the expected two copies. Most eukaryotic species are diploid, meaning they have two sets of chromosomes, whereas prokaryotes are haploid, containing a single chromosome in each cell. Aneuploids possess chromosome numbers that are not exact multiples of the haploid number and polysomy is a type of aneuploidy. A karyotype is the set of chromosomes in an organism and the suffix -somy is used to name aneuploid karyotypes. This is not to be confused with the suffix -ploidy, referring to the number of complete sets of chromosomes.

<span class="mw-page-title-main">Medical genetics</span> Medicine focused on hereditary disorders

Medical genetics is the branch of medicine that involves the diagnosis and management of hereditary disorders. Medical genetics differs from human genetics in that human genetics is a field of scientific research that may or may not apply to medicine, while medical genetics refers to the application of genetics to medical care. For example, research on the causes and inheritance of genetic disorders would be considered within both human genetics and medical genetics, while the diagnosis, management, and counselling people with genetic disorders would be considered part of medical genetics.

<span class="mw-page-title-main">Genetic analysis</span>

Genetic analysis is the overall process of studying and researching in fields of science that involve genetics and molecular biology. There are a number of applications that are developed from this research, and these are also considered parts of the process. The base system of analysis revolves around general genetics. Basic studies include identification of genes and inherited disorders. This research has been conducted for centuries on both a large-scale physical observation basis and on a more microscopic scale. Genetic analysis can be used generally to describe methods both used in and resulting from the sciences of genetics and molecular biology, or to applications resulting from this research.

In molecular biology, SNP array is a type of DNA microarray which is used to detect polymorphisms within a population. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. Around 335 million SNPs have been identified in the human genome, 15 million of which are present at frequencies of 1% or higher across different populations worldwide.

Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.

The following outline is provided as an overview of and topical guide to genetics:

A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.

Virtual karyotype is the digital information reflecting a karyotype, resulting from the analysis of short sequences of DNA from specific loci all over the genome, which are isolated and enumerated. It detects genomic copy number variations at a higher resolution for level than conventional karyotyping or chromosome-based comparative genomic hybridization (CGH). The main methods used for creating virtual karyotypes are array-comparative genomic hybridization and SNP arrays.

<span class="mw-page-title-main">Copy number analysis</span> Process of analyzing data produced by a test for DNA copy number variation

Copy number analysis is the process of analyzing data produced by a test for DNA copy number variation in an organism's sample. One application of such analysis is the detection of chromosomal copy number variation that may cause or may increase risks of various critical disorders. Copy number variation can be detected with various types of tests such as fluorescent in situ hybridization, comparative genomic hybridization and with high-resolution array-based tests based on array comparative genomic hybridization, SNP array technologies and high resolution microarrays that include copy number probes as well an SNPs. Array-based methods have been accepted as the most efficient in terms of their resolution and high-throughput nature and the highest coverage and they are also referred to as virtual karyotype. Data analysis for an array-based DNA copy number test can be very challenging though due to very high volume of data that come out of an array platform.

Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/neu oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH.

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.

Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional FISH methodology. In Q-FISH, the technique uses labelled synthetic DNA mimics called peptide nucleic acid (PNA) oligonucleotides to quantify target sequences in chromosomal DNA using fluorescent microscopy and analysis software. Q-FISH is most commonly used to study telomere length, which in vertebrates are repetitive hexameric sequences (TTAGGG) located at the distal end of chromosomes. Telomeres are necessary at chromosome ends to prevent DNA-damage responses as well as genome instability. To this day, the Q-FISH method continues to be utilized in the field of telomere research.

<span class="mw-page-title-main">9q34.3 deletion syndrome</span> Medical condition

9q34 deletion syndrome is a rare genetic disorder. Terminal deletions of chromosome 9q34 have been associated with childhood hypotonia, a distinctive facial appearance and developmental disability. The facial features typically described include arched eyebrows, small head circumference, midface hypoplasia, prominent jaw and a pouting lower lip. Individuals with this disease may often have speech impediments, such as speech delays. Other characteristics of this disease include: epilepsy, congenital and urogenital defects, microcephaly, corpulence, and psychiatric disorders. From analysis of chromosomal breakpoints, as well as gene sequencing in suggestive cases, Kleefstra and colleagues identified EHMT1 as the causative gene. This gene is responsible for producing the protein histone methyltransferase which functions to alter histones. Ultimately, histone methyltransferases are important in deactivating certain genes, needed for proper growth and development. Moreover, a frameshift, missense, or nonsense error in the coding sequence of EHMT1 can result in this condition in an individual.

The 2000s witnessed an explosion of genome sequencing and mapping in evolutionarily diverse species. While full genome sequencing of mammals is rapidly progressing, the ability to assemble and align orthologous whole chromosomal regions from more than a few species is not yet possible. The intense focus on the building of comparative maps for domestic, laboratory and agricultural (cattle) animals has traditionally been used to understand the underlying basis of disease-related and healthy phenotypes.

Nablus mask-like facial syndrome is a rare genetic condition. It is a microdeletion syndrome triggered by a deletion at chromosome 8 q22.1 that causes a mask-like facial appearance in those affected. This syndrome typically presents itself in infants, specifically newborns.

The Cancer Genome Anatomy Project (CGAP), created by the National Cancer Institute (NCI) in 1997 and introduced by Al Gore, is an online database on normal, pre-cancerous and cancerous genomes. It also provides tools for viewing and analysis of the data, allowing for identification of genes involved in various aspects of tumor progression. The goal of CGAP is to characterize cancer at a molecular level by providing a platform with readily accessible updated data and a set of tools such that researchers can easily relate their findings to existing knowledge. There is also a focus on development of software tools that improve the usage of large and complex datasets. The project is directed by Daniela S. Gerhard, and includes sub-projects or initiatives, with notable ones including the Cancer Chromosome Aberration Project (CCAP) and the Genetic Annotation Initiative (GAI). CGAP contributes to many databases and organisations such as the NCBI contribute to CGAP's databases.

Spatial transcriptomics is a method for assigning cell types to their locations in the histological sections. Recent work demonstrated that the subcellular localization of mRNA molecules, for example, in the nucleus can also be studied.

References

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  2. ↑ Bigner SH, Schröck E (November 1997). "Molecular cytogenetics of brain tumors". Journal of Neuropathology and Experimental Neurology. 56 (11): 1173–81. doi: 10.1097/00005072-199711000-00001 . PMID   9370227.
  3. ↑ O'Connor C (2008). "Fluorescence In Situ Hybridization (FISH)". Nature Education. 1 (1): 171.
  4. ↑ Martin EA, McFerran TA, eds. (2017). Dictionary of Nursing. Oxford University Press.
  5. 1 2 "Cytogenetic testing | DermNet NZ". www.dermnetnz.org. Retrieved 2020-04-02.
  6. 1 2 Banerjee D (15 January 2013). Array comparative genomic hybridization : protocols and applications. New York: Humana Press. pp. 1–13.
  7. ↑ Pinkel D, Albertson DG (2005). "Comparative genomic hybridization". Annual Review of Genomics and Human Genetics. 6 (1): 331–54. doi:10.1146/annurev.genom.6.080604.162140. PMID   16124865.
  8. ↑ Trask BJ (October 2002). "Human cytogenetics: 46 chromosomes, 46 years and counting". Nature Reviews Genetics. 3 (10): 769–78. doi:10.1038/nrg905. PMID   12360235. S2CID   1127220.
  9. ↑ Lucito R, Healy J, Alexander J, Reiner A, Esposito D, Chi M, et al. (October 2003). "Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation". Genome Research. 13 (10): 2291–305. doi: 10.1101/gr.1349003 . PMC   403708 . PMID   12975311.
  10. ↑ Robson SC, Chitty LS, Morris S, Verhoef T, Ambler G, Wellesley DG, Graham R, Leader C, Fisher J, Crolla JA (February 2017). "Evaluation of Array Comparative genomic Hybridisation in prenatal diagnosis of fetal anomalies: a multicentre cohort study with cost analysis and assessment of patient, health professional and commissioner preferences for array comparative genomic hybridisation". Efficacy and Mechanism Evaluation. 4 (1): 1–104. doi: 10.3310/eme04010 . PMID   28182369.
  11. 1 2 Rao PH, Nandula SV, Murty VV (2007). "Molecular cytogenetic applications in analysis of the cancer genome". In Fisher PB (ed.). Cancer Genomics and Proteomics: Methods and Protocols. Methods in Molecular Biology. Vol. 383. Humana Press. pp. 165–85. doi:10.1007/978-1-59745-335-6_11. ISBN   9781597453356. PMID   18217685.
  12. ↑ Wan TS (2017). "Cancer Cytogenetics: An Introduction". In Wan TS (ed.). Cancer Cytogenetics. Methods in Molecular Biology. Vol. 1541. Springer New York. pp. 1–10. doi:10.1007/978-1-4939-6703-2_1. ISBN   9781493967018. PMID   27910009.
  13. ↑ Speicher MR, Carter NP (October 2005). "The new cytogenetics: blurring the boundaries with molecular biology". Nature Reviews Genetics. 6 (10): 782–92. doi:10.1038/nrg1692. PMID   16145555. S2CID   15023775.
  14. ↑ Balajee AS, Hande MP (December 2018). "History and evolution of cytogenetic techniques: Current and future applications in basic and clinical research". Mutation Research/Genetic Toxicology and Environmental Mutagenesis. In memory of Professor Adayapalam T Natarajan. 836 (Pt A): 3–12. doi: 10.1016/j.mrgentox.2018.08.008 . PMID   30389159. S2CID   53274124.
  15. 1 2 GenomeOC (2013-01-18). "Cancer Genome Characterization Initiative". Office of Cancer Genomics. Retrieved 2019-10-05.
  16. ↑ "GenomeOC Research". Office of Cancer Genomics. 2013-02-04. Retrieved 2019-10-05.
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