The practice of clinical genetics has evolved substantially in a short period of time. Spanning the discovery of the number of chromosomes in a cell to the first recognized chromosomal syndrome, as the related technology has progressed, so too has determination of numerous causes of different syndromes. Specifically, this technology has allowed for the detection of detailed variants in a particular cancer. Doing so has enabled the development of new treatments in both the germline and somatic realms. Herein, we will describe the technological impact of chromosomal microarrays and next-generation sequencing—two of the most frequently used molecular diagnostic methods—to help us portray the genome on a much smaller scale, to the building blocks of all: the As, Ts, Cs and Gs.
Chromosomal Microarray Basics
Genomic microarrays, also called chromosomal or cytogenetic/cytogenomic microarrays (CMAs), enable analysis of deletions and duplications down to the exon level, as well as detection of regions of homozygosity (ROH). Other arrays, such as expression arrays, are used to examine RNA expression within a cell or tissue to detect what genes are turned on or off during development, or during cell differentiation. This article focuses on CMAs used in the clinical laboratory working in conjunction with chromosome analysis and fluorescence in situ hybridization (FISH).
CMAs examine all 46 human chromosomes at once (without needing to culture cells) by using millions of probes to detect copy number changes as small as 5 to 10 kilobases of DNA. In addition, single nucleotide polymorphisms (SNPs) detect regions of homozygosity that may contain mutated genes. Loss of heterozygosity (LOH) occurs when one allele is mutated, and the normal allele is lost. Copy neutral LOH (CN-LOH) is a loss of all or part of a chromosome and doubling of the remaining homologous material. Because there is no loss of material this region is considered copy neutral.
Next-Generation Sequencing Basics
Next-generation sequencing (NGS), or massively parallel sequencing, is a method of simultaneously sequencing millions of fragments of DNA and/or RNA and allows for the analysis of the whole genome or targeted gene regions in multiple patients at one time. NGS allows for amplification of single bases thousands (even millions) of times in a relatively short time period. This methodology is largely used to detect single nucleotide variants (SNV), small insertions or deletions, fusions, or amplifications in both germline (inherited) and somatic (acquired) specimens.
Application of CMAs
Generally, CMAs are chips or slides containing thousands of probes in triplicate, to which single stranded DNA from the patient is added and then allowed to hybridize to its complimentary sequence. A computer scanner examines the chip for fluorescence signal to determine changes in copy number or ROHs. Using oligonucleotides, CMAs enable diagnosis of genetic conditions by detecting small duplications and deletions that are below the resolution of chromosome analysis and fluorescence in situ hybridization (FISH). This recent technology led to the discovery of new genetic disorders and has allowed for the refinement of the size of existing microdeletion and microduplication critical regions. As CMA technology has improved, SNPs have been added, which allow for the further detection and discovery of ROHs and imprinting.
CMAs are now a recommended first-tier diagnostic test for the evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies.1 They also are recommended for patients undergoing invasive prenatal diagnosis when major fetal structural abnormalities are detected by ultrasound and when there is an intrauterine fetal demise or stillbirth.2
Several years ago, researchers and then clinical laboratories began to use CMAs to assess hematological malignancies and solid tumors, which provided information in helping to determine the diagnosis, prognosis, and potential therapy for neoplastic disorders.2 Further, CMAs are used to detect copy number changes and copy neutral LOH in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) to help inform the management and potential treatment of these patients.3 There are similar advantages to using CMAs in the assessment of chronic lymphocytic leukemia (CLL),4 acute lymphoblastic leukemia/lymphoma (ALL),5 multiple myeloma, and solid tumors.6
One of the drawbacks of CMA is its inability to detect low level mosaicism, point mutations, small deletions, duplications and inversions, and balanced chromosomal abnormalities, such as translocations. Therefore, CMAs would not be the test of choice to determine if, for example, a parent is a carrier of a balanced rearrangement or if a translocation has occurred in a hematological malignancy. FISH and chromosome analysis remain the best options for detecting balanced rearrangements.
Application of NGS
In molecular diagnostics, NGS is utilized to interrogate the entire genome, the exome (only the protein coding genes), or a small, targeted panel of genes. This method can detect SNVs and small structural abnormalities, such as deletions or insertions; detect gene fusions, suggesting a potential translocation of chromosomal material; detect copy number changes if the appropriate analysis tools are in place; and depending on the type of panel used, can detect methylation patterns for specific genes.
There are two main types of NGS platforms that can be employed in the lab. The first involves sequencing by synthesis, which is performed via sequencers utilizing fluorescent detection with all four bases being added at one time.7 The second analyzer platform has a single base added in each round. When an added base is incorporated, a hydrogen ion is released, accompanied by a pH change that is detected for each bead within a well; if a base is not incorporated, there is no voltage generated.7
Of course, there are advantages and disadvantages to each method. For example, the first series of analyzers mentioned above allows for paired-end reads (sequencing of both ends of the DNA or RNA fragment) but has a difficult time with GC–rich regions. Alternatively, the second series of analyzers mentioned were initially unable to perform paired end reads, but recent versions have succeeded in accomplishing this task. However, this method struggles with sequencing homopolymers (regions of the genome with highly repetitive sequence), as well as truncation errors.7 Thus, each lab must weigh the pros and the cons of each method depending on the intended goals of the technology and practice.
A Comparative Look
The overall process of NGS starts with DNA and/or RNA extraction (depending on what type of panel is being run), followed by quantification of specimen, library preparation, target enrichment, and finally sequencing. However, once the specimen is sequenced, the data needs to be analyzed. Due to this process resulting in massive amounts of data, a method of processing and organizing the data is necessary. A solid bioinformatics pipeline will help to remove the background noise produced by the instrument and help to remove any artifacts that may be introduced in the sample during preparation and/or sequencing. This can be a homebrew pipeline if the lab has bioinformaticians available, or it can be outsourced to a third party software that can process the data.
Given that multiple patient specimens can be run at one time on an NGS platform, each specimen requires a unique barcode to identify sequencing reads from specific specimens. Subsequently, the data from each of these individual specimens must be separated out (ie, demultiplexed). The resulting file is called the FASTQ file,8 which contains all the raw data from the assay. This FASTQ file is compared to the reference sequence in order to align the raw sequencing data and determine whether changes from the reference are present in the samples. This resulting file is the BAM file.9
Finally, once all the variant calls are made and the percentage of how frequently a particular variant is observed is determined, a variant call file (VCF) is created based on the BAM file. This file contains the quality analysis of the sequencing run, mapping of the reads to a reference sequence, and variant identification/annotation and frequency. From here, the interpretation component is performed:
Specific guidelines are available to aid in answering these questions, such as the ACMG/AMP guidelines for inherited syndromes and the AMP/ASCO/CAP guidelines for somatic changes (PMID: 25741868 and PMID: 27993330).
While the NGS method provides an enormous amount of information, drawbacks can include the high cost of reagents and the instruments themselves, the detailed validation process, the large amount of data generation (which then requires servers to store the data), and the complexity of the analysis, which may require molecular tumor boards to aid in interpretation. However, because of its acute level of sensitivity, it can detect tumor heterogeneity (different tumor cells can have different profiles between tumors and in the same tumor), tumor mutational burden (the total number of changes observed in tumor cells), and minimal residual disease (a very small amount of tumor cells left in the body after treatment). These can be substantial benefits, again, dependent on a specific lab’s clinical goals.
NGS and CMA have proven to be collaborative partners in aiding in the diagnosis and discovery of germline syndromes and somatic changes in cancers. For example, while NGS is getting better at detecting copy number variants, the use of CMA to detect these changes compliments the sequencing to give a broad picture of what may be occurring in the sample. Additionally, in some cancers such as brain tumors, the use of NGS to detect potential variants, fusions, or methylation patterns in combination with CMA to detect gains, losses, and loss of heterozygosity provides the entire picture to aid oncologists in determining what treatment or clinical trial a patient may be able to receive or participate in.
While both technologies have improved the detection rate of abnormalities in various diseases such as MDS, myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and myeloproliferative neoplasms (MPN),10 chromosome and FISH analyses are still invaluable tools for detecting balanced rearrangements, identifying individual clones, and clarifying complex clones, which aid in the determination of clonal evolution. In addition, FISH analysis can deliver a much faster turnaround time, which is essential for many diseases. Working together, these technologies in conjunction with real-time PCR, allow for a comprehensive picture of the state of disease and help determine the diagnosis, prognosis, and potential therapy for neoplastic disorders.
Virginia C. Thurston, PhD, FACMGG, is the director of the Parke Cytogenetics Laboratory at the Carolinas HealthCare System in Charlotte, North Carolina. The Parke Cytogenetics Laboratory performs chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray testing for over 50 hospitals. After earning her PhD from the University of Alabama at Birmingham, Jennie completed her clinical cytogenetics fellowship at Indiana University School of Medicine (IUSM). Following her fellowship, she joined the faculty and became the assistant director of the IUSM cytogenetics laboratory. While at IUSM, Jennie also was vice chair of education for the department of medical and molecular genetics, wherein she served as course director of medical genetics for the medical school and clinical cytogenetics for the graduate school. After leaving IUSM, Jennie established and directed the BayCare Cytogenetics Laboratory in Tampa, Florida.
Holli M. Drendel, PhD, FACMGG, is the director of the molecular pathology laboratory at Atrium Health, core laboratory in Charlotte, North Carolina. Before joining Atrium and Carolinas Pathology Group, she earned her PhD in medicinal chemistry and molecular pharmacology at Purdue University and completed fellowships in both molecular genetics and cytogenetics in the department of medical and molecular genetics at Indiana University School of Medicine.
Holli is board certified in both clinical cytogenetics and clinical molecular genetics by the American Board of Genetics and Genomics (ABGG). Following her fellowship, she moved to Fullerton Genetics at Mission Health in Asheville, North Carolina, where she directed the cytogenetics laboratory and expanded the test menu for both the molecular genetics and cytogenetics laboratories. Holli then moved to the Medical College of Wisconsin and codirected the Wisconsin Diagnostics cytogenetics laboratory. She is a member of several professional societies and her professional interests cover cytogenetics, FISH, molecular genetics, molecular oncology, and others.