Clinical drug response constitutes a complex phenotype emerging from the interplay of demographic, clinical, environmental, and genomic factors, among others. This complexity results in a marked interindividual variation in cardiovascular drug response, affecting both efficacy and toxicity, which can result in patient harm and the inefficient utilization of limited healthcare resources. Pharmacogenomics is the study and clinical application of the genetic determinants of drug response variation and aims to optimize drug efficacy and minimize adverse drug reactions through genotype-informed prescribing and monitoring recommendations. Pharmacogenomic associations have been established for several licensed cardiovascular drugs, including clopidogrel (CYP2C19), warfarin (VKORC1, CYP2C9, CYP4F2), and simvastatin (SLCO1B1). However, no cardiovascular pharmacogenomic association has yet been translated into routine clinical practice, which is attributable to a variety of reasons including logistical, financial, and knowledge barriers. Polygenic risk scores, multiomics approaches, and quantitative modeling are expected to become increasingly important in the effort to realize cardiovascular personalized medicine.
Human diseases reflect a spectrum of pathologies and mechanisms of disease pathogenesis. Despite the grouping of diseases according to their common features, the pathogenesis of various diseases is unique, and in some cases, multiple distinct mechanisms can give rise to a similar pathology (disease manifestation). Disease causation may be related to intrinsic factors or extrinsic factors, but many/most diseases are multifactorial, involving a combination of intrinsic (genetic) and extrinsic factors (exposures). It is now well recognized that most major diseases are ultimately the result of aberrant gene expression, and that susceptibility to disease is significantly influenced by patterns of gene expression in target cells or tissues for a particular type of pathology. It follows that gene mutations and other genetic (or epigenetic) alterations are important in the pathogenesis of many human diseases. Hence, molecular diagnostic testing for genetic alterations may (1) facilitate disease detection, (2) aid in disease classification (diagnosis), (3) predict disease outcomes (prognostication), and/or (4) guide therapy.
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with high mortality. Patients with a disease limited to the pancreas have a chance for a cure, but the likelihood of remission decreases if the cancer involves adjacent vessels or lymph nodes. Eventually the disease progresses to a terminal stage when the cancer is locally advanced and unresectable or when there are distant metastases. To prevent progression, the current goal is to detect PDAC in its earliest stages. Diagnostic biomarkers for pancreatic cancer must fulfill this criterion with a high sensitivity, specificity, and accuracy, but also be able to distinguish PDAC from other pancreatic conditions. Some of these conditions are associated with an increased risk of PDAC, but do not have the invasive disease at the time of identification. Although genetic alterations are well described in chronic pancreatitis, mucinous cystic neoplasms, and intraductal papillary mucinous neoplasms, the natural progression of these lesions to PDAC is not well understood. Over the last three decades the search for adequate diagnostic biomarkers for PDAC has closely followed the establishment of new biotechnological methods. Early methods detected protein biomarkers using enzyme-linked immunosorbent assay and Western blots. Subsequently the PCR was developed and found to have utility for the detection of genetic alterations. The latest chromatographic methods, gene-chips, and protein-chips enable a very broad search for diagnostic biomarkers including genome-wide screening. Most recently, epigenetic changes were found to be significantly altered in PDAC. Several studies have shown that panels of biomarkers increase sensitivity, specificity, and diagnostic accuracy in the diagnosis of PDAC, reflecting the fact that PDAC exhibits significant heterogeneity. Despite similar anatomic location cancers can have different biological backgrounds reflecting distinct molecular pathways to tumorigenesis, and therefore require different diagnostic biomarkers.
Glioblastoma (GBM) is the most common primary malignant brain tumor (WHO grade 4) with an overall survival following diagnosis of 1–2 years. GBM makes up approximately 12%–15% of all intracranial tumors and the great majority (60%–75%) of astrocytic tumors. Given the dismal prognosis for this malignancy, there has been an emphasis on characterizing the molecular alterations in these tumors for both prognosis and therapy. GBM development occurs due to alterations in several distinct pathways involving a combination of overexpression of growth factors and loss of apoptosis activators. With the explosion of molecular testing, particularly The Cancer Genome Atlas, GBM, as well as other types of gliomas, such as astrocytomas and oligodendrogliomas, are now characterized by distinct mutations in genes such as IDH and the presence or absence of chromosomal abnormalities such as the loss of 1p and 19q. This wealth of data has raised the possibility of better and more accurate characterization of this tumor for diagnostic, prognostic, and predictive purposes.
Fetuses with trisomy 21, or Down syndrome, can be identified in the first or second trimester of pregnancy using a combination of serum markers in maternal serum and ultrasound findings. The sensitivity and specificity of these modalities are quite good, being in the range of 95%. Recently, several groups have begun to exploit the power of massively parallel sequencing (MPS) to identify prenatal aneuploidies in cell-free fetal DNA that is found in the maternal circulation. Although court rulings regarding the patent positions surrounding this technology have led to other entities being able to offer this form of testing, including academic institutions, four major companies were originally founded with prenatal detection of fetal chromosome abnormalities as their primary focus and most testing in the United States is performed through these companies. The sensitivity and specificity of detection for Down syndrome using MPS are markedly superior to serum and ultrasound markers, being greater than 99%. However, it is important to stress to women seeking testing that the specificity of the MPS approach, while very high, is not perfect. This is a screening test and positive results need confirmation with invasive testing (amniocentesis). In this chapter, we review the techniques that each of these major, currently active U.S. companies are using for noninvasive prenatal screening.
The coronavirus disease-2019 (COVID-19) pandemic unquestionably presents an era-defining challenge to public health, social equilibrium, and the global economy. It highlighted numerous systemic deficiencies in the healthcare system of developing and developed countries, exposing the limitations of health facility preparedness for emerging infectious disease crises. Despite the unique challenges presented by a deadly virus, advancements in molecular diagnostics and vaccine development pipelines have dramatically reduced the potential response time in which these technologies can be integrated into public health efforts to minimize disease transmission and mortality. The territorial impact in managing the crisis was evident as we saw global disparities where some countries were affected more than others. In the United States, the lack of a coordinated response plan for a pandemic of this nature resulted in numerous diagnostic challenges in the approval and dissemination of molecular and serological tests on the national level. This chapter reviews how molecular and serological testing has been applied to the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as the regulatory and logistical hurdles that impeded the implementation of timely diagnostic testing. Additionally, monitoring the genomic surveillance of SARS-CoV-2 has augmented our understanding of viral evolution and persistent spread around the globe. The utilization of innovative molecular technologies allowed for more rapid development of efficient SARS-CoV-2 vaccines, albeit with inefficient dissemination and unequal availability on a global scale. The chapter will endeavor to highlight lessons learned from events in the COVID-19 pandemic, and how these lessons might improve the speed and efficiency with which nations are able to develop, validate and disseminate technologies for the diagnosis and control of novel pathogens in future pandemics.
The diagnosis and management of lung cancer today has been revolutionized by advances in molecular pathology and diagnostics. The discovery of oncogenic driver mutations in epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) genes and the development of targeted tyrosine kinase inhibitors have provided novel therapies that are the epitome of personalized medicine. There is a growing number of targetable genetic alterations, including rearrangements of ROS1 receptor tyrosine kinase (ROS1), Neurotrophic tyrosine receptor kinase (NTRK) and RET (RET), BRAF V600E and KRAS G12C mutations, as well as exon14 splice site mutations and amplification of MET (MET). The unique challenges in lung cancer diagnosis surround the optimal use of small-volume tissue samples for both pathological diagnosis and molecular analysis. This has encouraged the discovery of new molecular targets and technological innovations in molecular diagnostics that push the field forward. These have provided new hope for patients with lung cancer, which has traditionally been considered an incalcitrant cancer with limited effective therapies.
Spinal muscular atrophy (SMA) is characterized by progressive muscular weakness and atrophy. Most cases of SMA are caused by the homozygous deletion of the SMN1 gene. SMA has an incidence of 1/5,000–1/10,000, with a carrier frequency between 1/40–1/60. Carrier screening and newborn screening have become standard practice in many states in the United States and countries in Europe. Early identification of SMA during the presymptomatic phase is important due to the recent advances in SMA therapeutics, such as the Food and Drug Administration–approved nusinersen. If initiated early, therapy can be administered prior to the onset of symptoms and have the most benefit to the patient. Genetic counseling in the prenatal and postnatal period now involves a thorough explanation of therapeutic options for SMA.
The human body has an innate ability to delicately balance bleeding and clotting. The most important elements include endothelial cells, platelets, and proteins that help to form and break down fibrin thrombi. Genes that encode for coagulation proteins have been studied using DNA technologies to detect specific mutations that result in the formation of smaller amounts of protein or a molecule with less functional activity, which leads to a bleeding diathesis. Furthermore, overexpression of procoagulant proteins or a reduction in proteins with anticoagulant properties results in a clotting diathesis.
Renal cell carcinoma (RCC) represents a group of diseases arising from renal tubule epithelial cells and constitutes approximately 90% of all kidney cancers. The major socioeconomic impact of this disease continues to grow with its rising incidence worldwide. More than 76,000 new diagnoses of kidney cancer are projected in the United States in 2021, and an estimated 13,780 patient deaths will be attributed to this disease. The majority of patients present with organ-confined RCC, and surgical resection of these tumors has generally resulted in excellent long-term survival outcomes. However, it is reported that up to 30% of newly diagnosed patients present with nodal or distant metastasis. Systemic therapy has historically been reserved for patients in this setting with advanced or metastatic RCC.
Cancer is the leading cause of childhood death by disease after infancy, and its incidence, treatment, and outcome differ substantially from cancers in adults. Three of the most common childhood tumors are retinoblastoma, Wilms tumor (nephroblastoma), and neuroblastoma. These cancers arise from malignant transformation of developing cells (blasts) and are almost entirely restricted to the pediatric population. This chapter discusses the genetic basis of each of these tumor types, and the current role of molecular and cytogenetic testing in diagnosis, prognosis, and treatment. Retinoblastoma has a stable worldwide incidence of approximately 1/50,000 and accounts for 3% of all pediatric cancer diagnoses. The only known genetic locus affected in retinoblastoma is the RB1 gene at chromosome 13q14. Comprehensive molecular testing, including sequence analysis and deletion/duplication testing, has been demonstrated to identify mutation or deletion of RB1 in more than 95% of cases. Wilms tumor arises from nephrogenic rests of the developing kidney and represents approximately 7% of pediatric cancer diagnoses. An increased risk for Wilms tumor is associated with a number of predisposition syndromes, including Wilms tumor-aniridia-genitourinary abnormalities-intellectual disability, Denys-Drash syndrome, Beckwith-Wiedemann syndrome, Perlman syndrome, and Fanconi anemia subtypes D2 and N, among others. As the genetic etiology of the different syndromes varies, there are a large number of genetic loci underlying Wilms tumor predisposition. Molecular and cytogenetic testing is helpful for informing cancer risk and prognosis. Neuroblastoma, a solid tumor of the embryonic crest cells, accounts for approximately 10% of cancers but approximately 15% of cancer-related deaths among children. Genetic testing is useful for identifying germline mutations in predisposition syndromes, although these account for only 1%–2% of cases, and for identifying somatic alterations of prognostic significance. The best characterized acquired mutations include amplification of MYCN, ploidy status, and segmental chromosome abnormalities. Routine cytogenetic analysis and fluorescence in situ hybridization are essential for the proper characterization of neuroblastoma.
Respiratory tract infections (RTIs) are a spectrum of diseases associated with infection of both the upper and the lower respiratory tract. These diseases include the common cold, otitis media, influenza-like illness, croup, bronchiolitis, and pneumonia. Viruses are the most common cause of RTI and include influenza viruses, respiratory syncytial virus, human metapneumovirus, parainfluenza viruses, adenovirus, rhinoviruses, enteroviruses, and human coronavirus. These viruses have been responsible for two recent pandemics, the 2009 H1N1 influenza pandemic and the coronavirus disease 2019 (COVID-19) pandemic. It is often difficult to clinically differentiate viral and bacterial etiologies for some respiratory diseases. Nucleic acid amplification assays provide a rapid and extremely sensitive means to detect respiratory viruses. Multiplex and point-of-care molecular assays for respiratory viruses are widely utilized. Understanding the biology and pathogenesis of the associated viruses is key to understanding diagnostic testing limitations.
Spatial biology is emerging as a new field of research as technological advancements allow investigators to characterize high-plex profiles of RNA transcripts and proteins in the tissue isolated from patients or preclinical models. Spatial profiling is suitable for many applications, from discovery work through translational science, and may also have future utility in the clinic. Highly flexible tools have been developed that can be customized to answer a wide range of scientific questions in order to advance understanding of the fundamental biological processes that regulate health and disease. In this chapter, we provide an overview of one new spatial profiling platform, the GeoMx digital spatial profiler. This system is capable of quantitatively profiling protein and RNA expression for the investigation of molecular heterogeneity in the tissue microenvironment. It uses the morphology of biological structures within the tissue to direct a light beam to desired profiling regions, and the light cleaves photolabile barcoded profiling reagents from the surface of the tissue for downstream quantitation. The system is highly flexible with respect to the quantity and targets that can be profiled, as well as the definition of the profiling area. We briefly describe the principles of the assay, provide a primer on the technology, and show application examples from the literature of how the GeoMx platform is being used today. Finally, we present a forward-looking discussion of how the platform may be developed in the future to potentially enable clinical assays. Our goal is to provide information that will allow the reader to understand the strengths of the platform and appreciate the flexibility and utility of the system across a variety of research fields.
Next-generation sequencing (NGS) continues to push the boundaries of genomic medicine and transform clinical genome sequencing. With the introduction of NGS assays, whole-exome sequencing (WES) and whole-genome sequencing (WGS) are paving the way to interrogate many or all genes simultaneously as well as previously inaccessible portions of the genome. WES and WGS have integrated rapidly into molecular diagnostics with their clinical deployment being supported by increased automation that is tailored to both sample processing and sequencing as well as its downstream interpretation. With these advances comes an increasing need for high-quality bioinformatics that allows for careful variant interpretation, particularly as we begin to consider the relevance of noncoding variants to clinical diagnostic evaluations. This chapter takes a broad look at the scope of WES/WGS and its important role in molecular diagnostics.
Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and constitutes the sixth most commonly diagnosed malignancies with relatively low survival rate. Although complete resection remains the main treatment modality for early-stage HCC, many studies in the past several years have given us a better understanding of molecular pathogenesis and classification of HCCs, and novel molecular-based treatment and prognosis of HCC. This article serves as timely review of recent progress in molecular pathology of HCC.
Autism spectrum disorder (ASD) encompasses a heterogeneous group of conditions diagnosed by clinicians solely on the basis of behavioral assessments that reveal social communication deficits and repetitive behaviors. Individuals with ASD are increasingly being seen in clinical genetics. More than 100 genetic disorders that can exhibit features of ASD and dozens of rare susceptibility genes and copy number variation (CNV) loci have been identified, which combined, depending on the study, can facilitate a molecular diagnosis in ~5%–40% of ASD cases. So far, none of the known CNVs or genes account for more than 1% of ASD cases, and often different pathogenic variants are found within the same family. The likelihood of a genetic finding in ASD increases with the complexity of the phenotype (e.g., idiopathic or related to a specific genetic disorder or syndrome, with or without intellectual disability) and the technology used (e.g., karyotype, targeted gene, gene panel, microarray, exome sequencing, whole genome sequencing). The gene lists being used for clinical interpretation are also rapidly evolving. There are a growing number of examples of how defining genetic subtypes in autism may aid in early identification enabling earlier behavioral intervention, and informing prognosis, medical management, and assessment of familial recurrence risk. A workflow guiding genetic testing is presented.
