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Cytogenetics involves all aspects of chromosome biology. Through the application of molecular cytogenetic techniques in biology and medicine, we gain insights into the structural and functional organization of chromosomes, chromosomal abnormalities, and genomic variations in developmental, medical, and cancer genetics. This chapter explores recent advancements in molecular cytogenetics and their implications for managing hereditary diseases. It explores cutting-edge molecular methodologies such as spectral karyotyping, fluorescence in situ hybridization, and next-generation sequencing alongside the groundbreaking CRISPR genome editing technology. The chapter will highlight how these technologies have enhanced our understanding of chromosomal abnormalities, genome variation, and the genetic basis of hereditary diseases. Furthermore, it will shed light on the potential of these advancements in paving the way for novel therapeutic interventions aimed at rectifying genetic abnormalities associated with hereditary diseases, thereby offering new avenues for medical intervention and treatment.
Faculty of Sciences, Department of Biochemistry and Microbiology, Engineering and Agriculture, University of Venda, Thohoyandou, South Africa
Kachienga Leonard
Faculty of Sciences, Department of Biochemistry and Microbiology, Engineering and Agriculture, University of Venda, Thohoyandou, South Africa
Magwalivha Mpho
Faculty of Sciences, Department of Biochemistry and Microbiology, Engineering and Agriculture, University of Venda, Thohoyandou, South Africa
Nethathe Bono*
Faculty of Sciences, Department of Food Science and Technology, Engineering and Agriculture, University of Venda, Thohoyandou, South Africa
*Address all correspondence to: bono.nethathe@univen.ac.za
1. Introduction
Cytogenetics is a discipline of biology that focuses on the study of chromosomes and their heredity, particularly when applied to medical genetics [1]. The history of cytogenetics began with the description of thread-like structures in plant cell nuclei by Swiss botanist Nageli in the 1840s [2]. These structures are now known as chromosomes. These chromosomes become visible under a microscope during cell division, and specialized staining techniques enable the examination of their number and structure in diagnostic testing [2].
The science of cytogenetics is based on the genetics of cells whereby information from the parent material is passed to the offspring through hereditary [3]. This is determined by the functions and characteristics of the cell transmitted from parents to the offspring. This process applies to the germ cells of the ovary and testes, which, through fertilization become a new offspring and the somatic cells that produce offspring cells of the same tissue or organ where they reside [3]. Li and Pinkel [4] also defined cytogenetics as the relationship between aberrations (defects) in chromosomes and human genetic diseases. Furthermore, this area of genetics also enables the study of fundamental processes. One of them is the nature of inherited syndromes. These genomic alterations are involved in tumorigenesis, the whole genome of organisms or humans in a three-dimensional organization and epigenetic features of higher-order chromatin structure [4, 5]. Cytogenetics includes all aspects of chromosome biology as well as the use of molecular cytogenetic techniques in biology and medicine, such as chromosome structural and functional organization, genome variation, expression and evolution, chromosome abnormalities, and genomic variations in medical genetics and tumor genetics.
In clinical practice, cytogenetics plays a crucial role in evaluating infants and children with birth defects, investigating individuals experiencing infertility or recurrent pregnancy loss, analyzing prenatal and pre-implantation samples for chromosomal abnormalities, and identifying cancer-related chromosomal alterations in patients with solid tumors and hematologic neoplasms [6]. These same methodologies are also employed in cytogenetic research focusing on speciation, genome stability, and chromosomal evolution.
Molecular cytogenetic techniques are a vital component of the diagnostic process of genetic disorders caused by chromosomal abnormalities [6]. These techniques have been developed to get beyond the drawbacks of traditional cytogenetics, namely its poor resolution and incapacity to detect specific chromosomal abnormalities. Molecular cytogenetics includes various techniques briefly discussed below such as fluorescence in situ hybridization (FISH), multicolor FISH (M-FISH, SKY, and CCK), primed in situ labeling (PRINS), next-generation sequencing, spectral karyotyping and comparative genomic hybridization (CGH) [6].
2.1 Spectral karyotyping (SKY)
SKY is a molecular cytogenetic process that utilizes differential visualization techniques of all the chromosomes of human or organisms through image exposure with distinct colors within a single hybridization [7, 8, 9, 10]. It is also a process of staining chromosomes to analyze the chromosome complement of an organism, including the structure and number of chromosomes. The chromosome staining is done by the banding technique, which helps to visualize the dark (gene-rich) and light (gene-poor) regions of the chromosome [11]. SKY has been developed to clearly show and identify all 24 human chromosomes at once without requiring prior knowledge of any abnormalities involved. SKY can discern the abnormalities that cannot be detected by traditional banding techniques and fluorescent in situ hybridization (FISH). Making SKY a hypersensitive, hyper-accurate, and hyper-intuitionist method [12].
This technique also uses an integration of Fourier spectroscopy with epifluorescence microscopy with charged-coupled device imaging, according to Garini et al. [13], where probes are PCR-labeled through the incorporation of either haptenized or directly labeled nucleotides as a single reaction as dUTP labeled. These specific probe chromosomes are pooled together at the same time, and repetitive sequences are suppressed with excess cot-1 DNA in the hybridization mixture onto metaphase chromosomes. The hybridized chromosomes are then visualized using Fourier spectroscopy with epifluorescence microscopy with charged-coupled device imaging that allows triple bandpass to filter and permits simultaneous excitation of all the fluorochromes and the entire emission spectrum of metaphase in the range of between 400 and 800 nm [10]. Once a single image containing the spectra information for each image is obtained, the fluorescent intensities in the green, red and near infrared emission range are visualized in a standard display of red, green, and blue (RGB) [7]. The authors further opined that the pixels with the same spectral information are assigned a pseudocolor, which allows for the spectral classification of all chromosomes and the chromosomes are classified and aligned in a karyotype table. Interpretation and comparison of all aberrations are summarized in the karyogram.
It is used to detect inter-chromosomal structural aberrations (translocations and insertions) which are associated with balanced and imbalanced arrangements. This technique can be applied in the identification of cryptic translocation and expounding on the complex aberrations [14, 15, 16]. It is also used to identify various markers and ring chromosomes which are not unidentifiable by conventional banding techniques [17, 18, 19]. Intrachromosomal alterations resulting in small deletions or duplications and para- or pericentric inversions do not result in changes in chromosome size or spectral signature of the aberrant chromosome and, therefore, cannot be detected by SKY. Very small marker chromosomes or double minute chromosomes cannot always be unambiguously classified.
2.2 Fluorescence in situ hybridization (FISH)
This technique was first used to map the human genome and to localize a specific gene on a chromosome. It is further used in the clinical field to detect chromosomal aberrations such as duplication, deletion, balanced/unbalanced translocations, and monosomy/trisomy [11]. FISH is based on the use of chromosome region-specific and fluorescent-labeled DNA probes. FISH refers to the use of labeled nucleic acid sequence probes for the visualization of specific DNA or RNA sequences on mitotic chromosome preparations or in interphase cells [10]. Pinkel et al. [20, 21] developed a technique to visualize chromosomes using fluorescent-labeled probes called fluorescent in situ hybridization (FISH). The technique is the most convincing technique for locating the specific DNA sequences, gene mapping, diagnosis of genetic diseases, and genetic aberrations aiding various types of cancers or identification of novel oncogenes. FISH involves the annealing of RNA or DNA probes attached to a fluorescent reporter molecule with a specific target sequence of sample DNA, which can be viewed under fluorescence microscopy. The technique has advanced; through multicolor whole chromosome probe techniques such as spectral karyotyping, multiplex FISH, or an array-based method using comparative genomic hybridization, it is possible to screen the whole genome simultaneously. FISH has been acknowledged as a dependable diagnostic and discovery tool in the fight against genetic diseases and has transformed the field of cytogenetics [22]. The following processes or steps are involved: (a) double-stranded chromosomes, preferably of metaphase, are denatured (on fixed slides) by using formamide and heat, (b) the slide is pooled with fluorescent-labeled probes at 37°C, (c) fluorescent probes anneal at the complementary site of the chromosomes, and (d) visualize the slide under the fluorescence microscope [23].
2.3 Comparative genomic hybridization (CGH)
This process utilizes the reference DNA together with labeled tumor through hybridization on the normal metaphase chromosomes to generate a profile of DNA copy in tumor genomes along the chromosome length [10]. It is an important global screening that tests the aberrations in the chromosomes within a given tumor genome. According to Dorritie et al. [10], this process allows for the retrospective identification of aberrations within correlations of cytogenetics and prognosis. The most important thing about this process is that it requires less DNA and has proven useful in establishing a solid tumor progression in phenotype/genotypic [24]. The only drawback to this process is that it does not capture any information regarding the nature of chromosomal segments involved in a copy number of alteration (chromosome rearrangement).
Comparative genomic hybridization (CGH) is a unique FISH method (dual probes) used to identify any genetic abnormalities. The basis of the technique is comparing the total genomic DNA of a particular sample (such as tumor DNA) with the total genomic DNA of normal cells. Usually, two distinct fluorescent dyes are used to mark an identical amount of both tumor and normal DNA. This combination is then added and hybridized to a normal lymphocyte metaphase slide [25]. A fluorescent microscope equipped with a CCD camera and an image analysis system is used to view the slides, and evaluation software is used to count copies of genetic materials.
2.4 Primed in situ labeling (PRINS)
Primed in situ labeling (PRINS) is a versatile and inexpensive technique that has been used traditionally to identify tandemly repeated target sequences in chromosomes and nuclei as an alternative to FISH. The technology has the benefit of using very small probes that can quickly penetrate practically any target and can differentiate among closely similar DNA sequences in situ. This unit covers both the standard PRINS and the dideoxy-PRINS variant, which can significantly boost the reaction’s sensitivity [26].
In this throughput, metaphase chromosomes are used in the comparative genome hybridization, which is substituted by a high-density DNA microarray sported with artificial human bacterial chromosome (ABC) cloned [27]. They include cDNA sequences/PCR, degenerated primer PCR, or, for better genomic resolution, rolling circle amplification, which is incorporated [28]. Single nucleotide polymorphism (SNP) is used to assist in detecting rare and common genomic arrangements. This technique requires a single hybridization onto one array common genome hybridization process that is pegged on co-hybridization with a test together with a reference DNA [28].
3.2 Next-generation sequencing (NGS)
This technique is also known as second-generation or parallel sequencing. This approach entails arrays of several thousands of sequences in templates in parallel, generating numerous more copies of short reads of DNA per lane [29, 30]. Most of the common platforms used in this process are the following: 454 (Roche Applied Science, Branford, CT), Solexa (Genome Analyzer, Illumina Inc. Sandiego, CA), SOLiD (Applied Biosystems, Foster City, CA), Polonator (Dover/Harvard Systems, Salem, NH), and HeliScope (Helicos BioSciences, Cambridge, MA) [28]. The authors further reported that the NGS technique has been applied wholesomely in characterizing populations of expressed genes, all protein-coding genes (exomes), as shown in Figure 1. NGS also involves fragmentation of the DNA, immobilization of the fragments, and ligation of adapters to form libraries [28]. It is currently used in cancer research to establish disease-causing mutations and drug discovery because of its cost-effective and high sequencing capacity.
Figure 1.
Molecular cytogenic techniques and recent high throughput [28].
Molecular cytogenetics plays a vital role in the characterization and detection of chromosomal abnormalities, which are changes in the number or structure of chromosomes that can result in a variety of genetic disorders and diseases. To discover different chromosomal abnormalities, the area of molecular cytogenetics has greatly evolved, including cutting-edge techniques like karyotyping, gene rearrangement investigations, and molecular cytogenetics [31]. There are two types of chromosomal abnormalities: structural and numerical variations. Changes in chromosomal numbers, such as polyploidy (having multiple copies of the entire set of chromosomes) and aneuploidy (having extra or missing chromosomes), are examples of numerical variations. Conversely, structural variants refer to alterations in the chromosomal structure, including translocations, inversions, duplications, Insertion, Ring chromosomes, deletions, and cross-displacement (Figure 2).
Figure 2.
A schematic picture represents the chromosomal abnormalities. Chromosomal abnormalities can be divided into deletion, duplication and inversion, which occur in single chromosome (a), translocation (b) insertion (c) ringing the chromosome (d) and cross-displacement between two chromosomes (e). Picture obtained from Montazerinezhad et al. [31].
4.1 The detection and characterization of diseases resulting from chromosomal abnormalities
The detection and characterization of chromosomal abnormalities and the specific role of molecular cytogenetics in understanding structural variations are discussed below:
4.1.1 Hematologic malignancies
These are oncogenes that have an impact on immune systems such as blood, leukemia, lymph nodes, lymphoma and bone marrow. Combined analysis of FISH-based methods and karyotyping has been useful in the precise delineation of the karyotypes and accurate chromosome analysis of complex diagnostic cases. According to Das and Tan [28], with the introduction of high throughput genomic technologies such as NGS, FISH-based chromosome level detection has gradually changed focus to genome-wide detection of single nucleotide and indel variants that are common in cancer.
4.1.2 Gliomas
These are frequent tumors that occur in the central nervous system, and they are classified based on the cell type or non-neoplastic glial cells (ependymomas, astrocytomas, oligodendrogliomas and mixed gliomas such as oligoastrocytomas) on the supratentorial and infratentorial of the membrane of brain. The initiation of gliomas is based on multiple genetic alterations. The FISH technique is used to detect oligodendrogliomas which is associated with better response to adjuvant therapy because of imbalance translocation between chromosomes 1 and 19 [27]. The evaluation of the molecular status of each of these markers is a major relevant diagnostic and prognostic tool for brain tumors and is used by neuropathologists in routine clinical practice.
4.1.3 Sarcomas
These oncogenes constitute approximately 1% of all solids tumors, and there is considerable morphological overlap between various histogenetic types. Chromosomal karyotyping is generally the classic approach for identifying genetic markers and translocations in soft tissue sarcomas.
4.1.4 Carcinomas
Carcinomas, too, have highly complex karyotypes like sarcomas and the enumeration of these aberrations by molecular cytogenetics has rapidly increased.
Table 1 summarizes the detection and characterization techniques for the above-mentioned diseases and their advantages and disadvantages.
Method
Techniques
Application
Advantage/disadvantage
Cytogenetics
Karyotype
Detecting numerical and gross structural aberrations
Low resolution, time-consuming, and labor requirements
FISH
Detecting trisomies, monosomies and microdeletions
Detects mosaicism
CGH
Detects copy number variations of genetic material
Used only for losses and gains
Molecular
RFLP
Restriction fragments are separated by electrophoresis
Requires mutation in restriction site
ARMS PCR
Allele-specific amplification of mutant and normal allele, determination of the genotype of an individual
Highly sensitive possible to detect any known mutation May increase time and costs
Multiplex PCR
Amplification of more than one target simultaneously
Reduces time and labor requirements, lower sensitivity and specificity
Nested PCR
Amplification using external and internal primer sets
More sensitive, decreases nonspecific amplification
RT-PCR
Amplification of RNA
Amplification of all RNA types May increase time and costs
Real-time PCR
Amplification, detection, and quantification of target
Increased specificity Usually eliminates postamplification analyzes More expensive
MLPA
Deletions and duplications
A multiplex technique Identifies very small single gene aberrations (50–70 nt)
DGGE
Based on migration within gradient gel electrophoresis
Detects close to 100% of point mutations
SSCP
Based on migration within gel electrophoresis
Detects about 80–90% of point mutations
Heteroduplex analysis
Based on homoduplexes and heteroduplexes motilities in gel electrophoresis
Detects nearly 80% of mutations
CCM
DNA: DNA or DNA: RNA heteroduplexes are cleaved by piperidine
All possible mutations are detectable, uses toxic substances
PTT
It is based on a combination of PCR, transcription, and translation
Detects translation-terminating mutations, missense mutations are not detected
OLA
It is based on the ligation of two flanked primers annealed with target sequences
Detects all base exchanges
Table 1.
Cytogenetics and molecular methods for mutation detection [25].
FISH: fluorescence in situ hybridization; CGH: comparative genomic hybridization; RELP: restriction fragment length polymorphism; ARMS; amplification refractory mutation system; PCR: polymerase chain reaction; RT: reverse transcriptase; MLPA; multiplex ligation-dependent probe amplification; DGGE: denaturing gradient gel electrophoresis; SSCP: single strand conformational polymorphism; CCM: chemical cleavage of mismatch; PTT: protein truncation test; OLA: oligonucleotide ligation assay.
The molecular cytogenetic techniques have significantly impacted the understanding of genome variations and genetic diseases. These techniques, such as fluorescence in situ hybridization (FISH), have enhanced the resolution and diagnostic utility of cytogenetic analysis, enabling the specific detection of unique sequences, chromosomal regions, or entire chromosomes in cells [22, 32]. They have played a crucial role in identifying disease-causing mutations and variants by providing insights into genomic variations in unaffected individuals and those with morbid conditions affecting all cells of an organism [6]. In the context of hereditary diseases, molecular cytogenetics has become a vital tool for diagnosing genetic disorders caused by chromosomal abnormalities. By combining cytogenetics and molecular biology, molecular cytogenetics has increased the resolution of cytogenetic analysis, allowing for the detection of subtle and gross chromosome rearrangements, heteromorphisms, and other genomic variations that can have diverse phenotypic effects [6]. These techniques have revolutionized the field by enabling the study of fundamental biological questions related to inherited syndromes, tumorigenesis, and the three-dimensional organization of the human genome. By utilizing molecular cytogenetic approaches, researchers can identify chromosomal abnormalities with higher precision and delve into the genetic basis of hereditary diseases, ultimately aiding in the identification of disease-causing mutations and variants that underlie various genetic disorders [6].
The potential of molecular cytogenetic advancements in therapeutics lies in the ability to correct genetic abnormalities through gene therapy approaches [33, 34]. Gene therapy involves altering the genetic code to restore the functions of critical proteins affected by genetic mutations. This can be achieved through gene transfer therapy, where a normal copy of the gene is introduced to replace a faulty one, or through genome editing, which modifies existing DNA in cells. The use of viral vectors, such as adenoviruses and lentiviruses, has been successful in delivering genetic material for gene therapy, although it carries some risks like triggering immune responses or causing errors leading to cancer [35, 36, 37]. Researchers are also exploring non-viral vector technologies, like nanoparticles, to deliver genetic material more safely and effectively. Challenges in gene therapy include ensuring safety and efficiency, and addressing the risks and benefits specific to each disease being treated [38]. The development of gene therapy requires careful consideration of factors like sustained manufacturing of therapeutic gene products without harmful side effects [37, 39]. Future directions in therapeutic interventions involve overcoming technical challenges, improving the delivery of genes or gene-editing tools to target specific cells, and enhancing control over the functionality and timing of treatments within the body. As gene therapy continues to evolve, advancements in vector technologies, delivery systems, and understanding of genetic diseases will pave the way for more effective and targeted therapeutic interventions.
The sources provided offer valuable insights into molecular cytogenetics and its clinical applications. Molecular cytogenetics, a field combining molecular biology and cytogenetics, focuses on analyzing chromosome structure to distinguish normal and cancer-causing cells. It plays a crucial role in diagnosing and treating various malignancies, including hematological malignancies and brain tumors. Techniques like fluorescence in situ hybridization (FISH) are commonly used to detect chromosomal abnormalities associated with genetic diseases and cancer [6]. In clinical settings, molecular cytogenetics is instrumental in diagnosing genetic disorders caused by chromosomal abnormalities. It has significantly advanced diagnostic capabilities beyond classical cytogenetics by increasing resolution and diagnostic utility. Techniques like FISH, multicolor FISH, and comparative genomic hybridization (CGH) are pivotal in identifying chromosomal abnormalities and understanding their diagnostic implications [40]. Case studies in molecular cytogenetics demonstrate its diagnostic and prognostic applications in oncology and medical genetics. By analyzing chromosome structure, these studies provide insights into the genetic causes of cancer, potential treatment strategies, and personalized medicine approaches based on cytogenetic findings. The field continues to evolve, with ongoing projects like the Cancer Genome Characterization Initiative (CGCI) focusing on genomic research for rare cancers to elucidate genetic abnormalities and their role in cancer pathogenesis [40].
The ethical considerations surrounding genome editing and genetic manipulation are a topic of significant debate and concern. Various international bodies and studies have addressed these issues, emphasizing the importance of ethical guidelines, transparency, responsible science, respect for persons, fairness, and transnational cooperation [41]. One key ethical concern is the distinction between somatic and germline interventions, as well as between therapy and enhancement. The difficulty in drawing clear lines between these categories has led to debates on the normative implications of human nature, consent from future generations, potential slippery slopes toward eugenics, and implications for justice and equality [42]. Regulatory frameworks play a crucial role in governing genome editing technologies. Oversight of human genome editing is embedded within the larger context of international conventions and norms for the protection of human rights and research involving human subjects. Principles for governance of genome editing have general application globally and are reflected in specific statutory and regulatory rules adopted by various nations [41]. Looking toward the future, it is essential to continue developing robust ethical guidelines, fostering public engagement and dialog, establishing global regulatory bodies, and promoting international collaborations to ensure that the use of gene-editing technologies is grounded in international ethical standards and addresses the ethical challenges associated with these advancements [43].
In conclusion, molecular cytogenetics continues to be a vital field in both biological and medical research, offering invaluable insights into the intricate world of chromosome biology. Through cutting-edge techniques such as spectral karyotyping, fluorescence in situ hybridization, next-generation sequencing, and CRISPR genome editing, researchers have made significant strides in understanding chromosomal abnormalities, genome variations, and the genetic underpinnings of hereditary diseases. The advancements discussed in this chapter not only deepen our comprehension of genetic disorders but also hold immense promise for clinical applications. The potential to correct genetic abnormalities through genome editing opens new avenues for medical treatment, offering hope to individuals affected by previously incurable conditions. The advancements in molecular cytogenetics outlined in this chapter have the potential to revolutionize the way we diagnose, manage, and treat hereditary diseases, heralding a new era of precision medicine.
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Written By
Rikhotso Mpumelelo Casper, Kachienga Leonard, Magwalivha Mpho and Nethathe Bono
Submitted: 11 June 2024Reviewed: 16 June 2024Published: 16 January 2025
Open access peer-reviewed chapter - ONLINE FIRST
