Molecular and genomic methodologies for clinicians

Over the last decade, molecular diagnostic testing in patients with hematologic and oncologic disorders has become increasingly sophisticated and prevalent. While in the past focused genetic tests were performed, in recent years the widespread use of genomic and molecular approaches in both research and clinical settings has refined diagnostics and therapeutics for pediatric blood disorders and cancer. This chapter provides an overview of the currently used molecular and genomic methods, with the goal of introducing these new technologies for trainees and clinicians without extensive laboratory experience. This is not meant to be a comprehensive review of the topic and will primarily focus on genetic methods that are currently used in clinical settings.

Clinical molecular and genomic methodologies: goals

The goal of genetic and genomic analysis is the identification of molecular lesions underlying patient disease and using the information to inform clinical care. These include:

1.
Identification of the causative gene or mutation, usually by sequencing a gene or panel of genes known to be associated with a specific disorder such as thrombocytopenia, thalassemia, or familial cancer predisposition syndromes.
2.
Evaluation of genomic or molecular markers in multiple affected and unaffected individuals (related or unrelated) through indirect approaches. These often identify markers that may be coinherited with a disease but do not cause the disease itself. Markers may be single nucleotide variants (SNVs) or structural variations such as insertions, deletions, and copy number variation.
3.
Genome-wide evaluation of genes and markers for the purpose of diagnosis of a disease or directing treatment, typically through the use of next-generation sequencing (NGS) technologies. These include whole exome and genome sequencing (WES and WGS) and are currently being applied increasingly for clinical applications in diagnostics and therapeutics.

Table 1.1 lists the commonly used genetic testing methodologies, along with the types of molecular lesions that they are able to identify.

Table 1.1

Overview of molecular and genomic diagnostic methodologies.

Modified from Sankaran, V. 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier.

Method	Common point mutations	Rare point mutations	Copy number variants	Uniparental disomy	Balanced inversions or translocations	Repeat expansions	Examples of use in pediatric hematology/oncology
Linkage analysis (using markers such as short tandem repeats)	X		X				Family pedigree with history of hereditary spherocytosis and interest in identifying the causal gene
Fluorescence in situ hybridization			X		X		Acquired monosomy in myelodysplastic syndrome
Array comparative genomic hybridization			X	X			Testing for microdeletion in patient with hematologic and syndromic phenotype
Genome-wide single nucleotide variant microarrays	X		X				Testing for small copy number variants in pediatric leukemia
Targeted polymerase chain reaction analysis	X	X				X	Testing for JAK2 V617F mutation in patient with a myeloproliferative disorder
Sanger sequencing	X	X					Molecular diagnosis of a patient with pyruvate kinase deficiency
Gene panel sequencing	X	X					Hematologic and solid tumors, severe congenital neutropenia
Whole genome or exome sequencing	X	X	X				Hematologic and solid tumors, unknown bone marrow failure syndrome

Methods of genetic analysis

Markers of genetic defects

While most methods are now focused on identifying the precise molecular cause of disease through sequencing, indirect tests have been quite useful in the past and are still used today, particularly for mapping causes of a disease in a family. These are found throughout the genome, include SNVs or short tandem repeats (two to five base long repetitive elements with varying numbers of repeats), and have been extremely useful as a way to identify likely causal genes. These markers may be located in close proximity to the causal mutation and thus within a family should only be found in affected members, suggesting that the causal mutation is located nearby. This approach, a process called linkage analysis , is particularly useful when causative mutations reside in regulatory regions such as enhancers or promoters, rather than protein coding sequences. Genome-wide single nucleotide polymorphism (SNP) microarrays accelerated the use of these approaches prior to large-scale sequencing. These methods have been used as an initial screen in large families or populations with suspected cancer predisposition syndromes to guide in-depth analysis of certain regions of the genome, prior to NGS being available. However, as costs decrease, genome-wide approaches such as NGS can be used initially, with efforts subsequently directed to linkage analysis if sequencing fails to detect the disease-causing mutation.

Other technologies are useful for assessing large-scale structural chromosome defects. Fluorescence in situ hybridization (FISH) is a cytogenetic technique that queries whether chromosomes or chromosomal fragments are duplicated or deleted and has been particularly useful for mapping gene locations and classifying malignant tumors. Polymerase chain reaction (PCR)-based methods have been used with increasing frequency as a replacement for FISH. Another method that has been highly used in recent years for examination of copy number variation and structural defects is array comparative genomic hybridization (CGH), although this may be supplanted by NGS. Despite the increased use of PCR and NGS, FISH remains one of the best clinically available methods to detect classic cytogenetic changes that are diagnostic and implicated in a number of pediatric cancers such as translocations that are frequently seen in leukemia and certain solid tumors.

As with sequencing (see later), all of these technologies may detect changes that are of unclear significance and should be interpreted with caution. Array genotyping technologies are most commonly used by direct-to-consumer services that report questionable relative risk information to individuals who request such services. However, as large-scale NGS has become more affordable, arrays have been used with decreasing frequency.

Sequencing approaches

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