Clinical Precision Medicine

A Primer

Judy S. Crabtree

• Publisher: Academic Press
• Year: 2019
• ISBN:
  9780128204023
• Topic: Medicine
• Subtopic: Genetics in Medicine

Clinical Precision Medicine: A Primer offers clinicians, researchers and students a practical, up-to-date resource on precision medicine, its evolving technologies, and pathways towards clinical implementation. Early chapters address the fundamentals of molecular biology and gene regulation as they relate to precision medicine, as well as the foundations of heredity and epigenetics. Oncology, an early adopter of precision approaches, is considered with its relationship to genetic variation in drug metabolism, along with tumor immunology and the impact of DNA variation in clinical care.

Contributions by Stephanie Kramer, a Clinical Genetic Counselor, also provide current information on prenatal diagnostics and adult genetics that highlight the critical role of genetic counselors in the era of precision medicine.

• Includes applied discussions of chromosomes and chromosomal abnormalities, molecular genetics, epigenetic regulation, heredity, clinical genetics, pharmacogenomics and immunogenomics
• Features chapter contributions from leaders in the field
• Consolidates fundamental concepts and current practices of precision medicine in one convenient resource

Chapter 1

Cytogenetics in precision medicine

Fern Tsien, PhD Associate Professor, Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, United States

Abstract

Cytogenetic analysis is a conventional technique utilized worldwide to diagnose chromosome instability and may indicate the presence of a genetic disorder or malignancy; historically, it was one of the first techniques utilized for precision medicine. The field of clinical human cytogenetics (the study of human chromosomes, their structure, inheritance, and abnormalities) intensified with the discovery of the normal human chromosome number of 46 in 1956 and of trisomy 21 in Down syndrome in 1957. The presence of a 9; 22 translocation found in a patient with chronic myelogenous leukemia in 1959 led to the development of therapies targeting the oncogenic fusion protein produced by the “Philadelphia chromosome.” Today, cytogenetic analysis continues to provide essential diagnostic, prognostic, and therapeutic information for cancer precision medicine and research.

Keywords

Chromosome; Cytogenetics; Double minute; FISH; G-banding; Homogeneously staining region; In situ hybridization; Instability; Karyotype; Translocation

1. Cytogenetic techniques
2. G-banding (karyotype) analysis
3. Chromosome abnormalities
4. Further reading

Cytogenetic techniques

Cytogenetic analysis traditionally involves G-banding (karyotyping). A higher resolution detection of constitutional and cancer-acquired chromosomal abnormalities can be achieved by combining the karyotype with molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) and microarray comparative genomic hybridization (aCGH). Each procedure has its advantages and limitations and can provide unique information regarding a patient's diagnosis and disease progression.

G-banding (karyotype) analysis

Karyotype analysis is highly efficient at identifying numerical chromosome abnormalities (e.g., trisomy, triploidy) and structural rearrangements (e.g., insertions, deletions, inversions, translocations) and is effective in uncovering cell population heterogeneity (Fig. 1.1). A limitation of this procedure is that aberrations ess than 1 Mb in size may be missed. Furthermore, to analyze metaphase chromosomes and identify rearrangements, living cells are required that are either actively undergoing cell division or induced to divide with the help of mitogens. Therefore, karyotype analyses cannot be performed on formalin-fixed paraffin-embedded (FFPE) tissue samples. Despite these limitations, G-banding is widely employed in both the research and clinical settings.

G-banding can be performed on almost any cell type that can be cultured (fresh live cells), including peripheral blood, solid tumors, bone marrow, skin fibroblasts, miscarriage material (products of conception), amniotic fluid, and chorionic villus sampling (CVS). Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. When a cell culture has reached an exponential phase with a high mitotic index, the cells are arrested at metaphase by disrupting the spindle fibers and preventing them from proceeding to the subsequent anaphase stage. The cells are treated with a hypotonic solution, preserved in their swollen state with a methanol-acetic acid fixative solution and then dropped onto glass microscope slides. The process of G-banding involves trypsin treatment followed by Giemsa staining to create characteristic light and dark bands.

Figure 1.1 Structural chromosome abnormalities.

Each individual chromosome can be identified by its distinct banding pattern and plotted on an ideogram or a map corresponding to the specific regions of each of the chromosomes. A classification system has been established in which each chromosome band is assigned a sequential number, starting from the centromere and increasing as one approaches the end of the telomere. All cytogenetic reports and publications utilize this International System for Human Cytogenetic Nomenclature (ISCN), which is continuously updated.

FISH is a procedure that combines basic principles of molecular biology and cytogenetics to evaluate chromosome abnormalities at a higher resolution than classic karyotyping. The procedure involves the hybridization, directly on the microscope slide, of a fluorescently labeled DNA probe to a complementary gene or chromosomal region. One of the main advantages of FISH is that it can be performed on mitotic and interphase cells, allowing for the analysis of archived tissue samples. Another benefit of FISH is that multiple probes of differing color can be implemented to concurrently analyze multiple genes, regions, or chromosomes, detecting translocations, amplifications, or other rearrangements diagnostic for a particular type of malignancy. FISH is ideally suited for the study of cancer-related chromosome instability (CIN), since it enables the analysis of cell morphology, and as a result, cell-to-cell heterogeneity. In general, both the number and size of FISH signals can be quantified, providing insight into the nature of a specific chromosomal aberration. One limitation of FISH is that the DNA probes relevant to a region of interest are not always commercially available. In addition to an ability to assess cell-to-cell heterogeneity, FISH can also evaluate CIN in samples isolated from the same patient at different time points to monitor disease progression and treatment response. Penner-Goeke et al. employed interphase FISH and assessed CIN in serial samples collected from women with ovarian cancer. They showed that an increase in CIN was observed in women with a treatment resistant form of the disease.

aCGH is a microarray procedure that can determine DNA sequence copy number changes throughout the entire genome. Fluorescently labeled DNA extracted from clinical samples is used as a probe. This DNA is mixed with normal labeled reference DNA and hybridized to a microarray chip. The laboratory utilizes specific computer software to view the ratio between the sample DNA (green) and the reference DNA (red), to determine gains or losses of DNA. Array CGH is used to detect amplifications, deletions, and chromosome gains and losses and is often implemented in cancer cytogenetic studies.

When aCGH is employed to compare the frequency of chromosomal imbalances in primary colorectal tumors and brain metastases, it can reveal a higher degree of sensitivity with regard to segmental aneuploidy in metastatic lesions. However, since aCGH employs pooled DNA samples isolated from large numbers of cells, it is incapable of measuring the level of cell-to-cell heterogeneity in chromosome number and structure that is characteristic of CIN. Another limitation of aCGH is that although it is efficient in detecting gains and losses of DNA material, it cannot detect rearrangements that involve inversions or translocations (i.e., the 9; 22 translocation in chronic myelogenous leukemia).

Chromosome abnormalities

Congenital chromosomal abnormalities usually result from abnormal nondisjunction or chromosome rearrangements during meiosis I or meiosis II (when the gametes are formed, prior to fertilization) and are sometimes parentally inherited. Alternatively, chromosome abnormalities may occur during the mitotic cell division of early development, resulting in mosaicism (two or more different cell populations). Chromosome abnormalities can be numerical (i.e., trisomy, monosomy, or polyploidy) or structural (i.e., deletions, duplications, inversions, insertions/substitutions, and translocations). In humans, chromosome abnormalities occur in approximately 1 per 160 live births, 60%–80% of all miscarriages, 10% of stillbirths, 13% of individuals with congenital heart disease, 3%–6% of infertility cases, and in many patients with developmental delay and/or other birth defects. Some disorders caused by congenital chromosome abnormalities can affect early prenatal development and may not be amenable to precision medicine.

However, germline chromosomal abnormalities or mutations can be associated with hereditary forms of cancer that may be responsive to precision medicine. For example, retinoblastoma patients who have the hereditary form (i.e., germline carriers of an RB1 mutation or a chromosome 13q14 deletion) also have a risk of developing secondary malignant neoplasms such as osteosarcomas, soft tissue sarcomas, and melanoma. This risk of malignancy is maximized by external beam radiotherapy treatments, which is why these treatments are now avoided for patients with the hereditary...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Contributors
• Chapter 1. Cytogenetics in precision medicine
• Chapter 2. Molecular genetics—the basics of gene expression
• Chapter 3. Fundamentals of epigenetics
• Chapter 4. Fundamentals of heredity
• Chapter 5. Clinical genetics
• Chapter 6. Pharmacogenomics
• Chapter 7. Immunogenomics: steps toward personalized medicines
• Chapter 8. Technology of clinical genomic testing
• Index