Cytogenetics and large scale structural genomic variation

3.1 Introduction

Structural genomic variation is an integral part of our variable genomes, both in health and disease. Structural variation has been defined as involving segments of DNA more than 1 kb in size. This ranges from very large 'microscopic' structural variants (visible down a microscope and typically more than 3 Mb in size) to smaller 'submicro-scopic' structural variation (Feuk et al. 2006a; Scherer et al. 2007). In this chapter the focus is on large scale microscopic structural variation, which was the subject of early research into human genetic diversity through cytogenetic approaches. As technology has advanced, our ability to resolve submicroscopic structural variation has dramatically improved, demonstrating that this class of structural genomic variation is much more common than previously thought (reviewed in Chapters 4 and 5). For both microscopic and submicroscopic structural variation, diversity may arise through copy number changes involving deletions, insertions, or duplications, or structural variation resulting in a positional change, for example balanced translocations, or an alteration in orientation, as seen with inversions (Fig 1.29) (Scherer et al. 2007).

In this chapter major classes of chromosomal variation are described ranging from gain or loss of whole chromosomes to specific chromosomal rearrangements and resulting structural variation such as deletions, duplications, inversions, and translocations. The impact of such diversity on human health is illustrated by discussion of a number of important associated clinical syndromes. For further information, a number of excellent reviews have been published on this subject (Section 3.7) and the interested reader is also referred to the diverse array of databases and other electronic resources that have been established to try and catalogue human structural variation and the phenotypes that may be associated with them (Box 3.1). A detailed description of terminology used to describe human chromosomes is also available, published as recommendations of the International Standing Committee on Human Cytogenetic Nomenclature (Shaffer and Tommerup 2005).

3.2 A historical perspective on cytogenetics

The study of human chromosome number and variations in chromosome structure has provided the basis for cataloguing and investigating human genetic diversity. Although human chromosomes were first described in the 1880s by Flemming and Arnold, it was only in 1956 that Tjio and Levan at the University of Lund in Sweden correctly reported that we possess 46 chromosomes within a normal cell rather than the long-held view that there were 48 (Fig. 3.1) (Tjio and Levan 1956). This work, combined with other conceptual and technological advances at the time, led to a very rapid period of development in the study of chromosomes and associated chromosomal abnormalities. This established human cytogenetics as a scientific discipline that has continued to play a key role in clinical medicine and the study of human genetics (Trask 2002). Some of the important discoveries and advances relating to cytogenetics are summarized in Fig. 3.2.

The careful association of chromosomal abnormalities with observed phenotypes in specific human disorders

Box 3.1 Electronic resources and databases of human structural genomic variation

A number of different databases and resources are available, in many cases including both microscopic and submicroscopic variation.

Large scale (microscopic) variation

• Chromosomal Anomaly Collection (www.som. Register/). Database including large scale copy number variation in phenotypically normal individuals. Unbalanced chromosomal abnormalities without apparent phenotype are described, including duplications and deletions. Also includes large scale cytogenetically visible copy number variants.

• Chromosome Abnormality Database (www.ukcad. Database containing constitutional and acquired abnormal karyotypes reported by UK regional cytogenetics centres.

• DECIPHER: DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources ( Database covering submicroscopic chromosomal imbalances (chromosomal microdeletions, duplications, insertions, translocations, and inversions). Clinical information and genomic location.

• ECARUCA: European Cy to geneticists Association Register of Unbalanced Chromosome Aberrations ( ecaruca.jsp). Database relating to rare chromosomal disorders including microdeletions and microduplications.

has proved extremely informative. In 1959, gain or loss of specific chromosomes was associated with a number of disorders, including Down syndrome due to possession of an extra copy of chromosome 21 (Lejeune et al. 1959), while gain of an X chromosome was found to cause Klinefelter syndrome (XXY) (Jacobs and Strong 1959), and its loss Turner syndrome (XO) (Ford et al. 1959). In

• Human Structural Variation Project (http://paralogy. gs This database catalogues large and intermediate scale structural variation together with copy number polymorphism.

• OMIM: Online Mendelian Inheritance in Man ( An extensive and frequently updated summary of human genes, genetic variation, and genetic disorders written and edited by Johns Hopkins University and distributed by the National Center for Biotechnology Information (NCBI) (Hamosh et al. 2005).

Smaller scale (submicroscopic) variation

• Copy Number Variation Project (www.sanger. Data for copy number variation within HapMap samples hosted at the Wellcome Trust Sanger Institute, UK.

• Database of Genomic Variants (http://projects. A catalogue for data from healthy control subjects of structural variation (greater than 1 kb in size) and indels (100 bp to 1 kb).

• dbSNP ( A catalogue of human genome variation maintained by the NCBI (Sherry et al. 2001).

• Human Gene Mutation Datatabase (www.hgmd. org). Database of mutations causing or associated with human inherited disease (Stenson et al. 2003). Restricted to germline mutations and nuclear genes.

1963 the first inherited syndrome due to a chromosomal deletion was reported named 'cri du chat', a syndrome characterized by affected babies having a high pitched, cat-like cry due to deletion of some or all of the short arm of chromosome 5 (Box 5.9) (Lejeune et al. 1963). It was also quickly appreciated that the majority of spontaneous abortions could be attributed to chromosomal

Chromosome Joe Hin Tjio
Figure 3.1 Human cells contain 46 chromosomes. Photomicrograph showing the normal human 46,XY chromosome number by Joe-Hin Tjio, December 1955. Reprinted with kind permission from Springer Science + Business Media: Human Genetics (Harper 2006).

abnormalities (Clendenin and Benirschke 1963), while development of amniocentesis allowed for the screening and identification of fetal chromosomal abnormalities. The key role of chromosomal defects in human cancer was also made clear by a number of studies, including the discovery in 1960 of the Philadelphia chromosome as a cause of chronic myeloid leukaemia (Nowell and Hungerford 1960), later shown to be the result of a translocation between chromosomes 9 and 22 (Rowley 1973). Here, as elsewhere in this book, our focus is on constitutional human genetic diversity, while detailed discussion of the essential and expanding role of cytogenetics in cancer is beyond the scope of this chapter.

A series of key technological developments have characterized advancement of the field of cytogenetics. Essential among these was the establishment of banding technologies such that not only could individual chromosomes be specifically identified, but also locations within them (Caspersson et al. 1968). Staining of chromosomes revealed dark and light bands, a 'barcode' with which deletions, inversions, insertions, and other rearrangements could be resolved and studied (Fig 1.2). This was later taken to progressively higher states of resolution. Approaches such as somatic cell hybridization allowed the mapping of genes and markers to specific chromosomes (Weiss and Green 1967; Ruddle et al. 1971). Flow cytometry and sorting proved to be very useful techniques in characterizing human chromosomal variation, both quantitatively and in separating normal and abnormal chromosomes (Carrano et al. 1979). Hybridization of specific labelled DNA probes proved highly informative, particularly after the development of fluorescent labels, which proved safer and to give higher resolution than radioactive labelling (Landegent et al. 1985). Fluorescence in situ hybridization (FISH) techniques have grown in sensitivity to remarkable levels and have been used in the discovery of a number of key chromosomal rearrangements including the remarkable imprinting disorders Prader-Willi (Box 5.6) and Angelman syndromes (Box 5.7) (Knoll et al. 1989). The development of chromosome-specific paints and use of a combination of fluorochromes has allowed multiplex FISH or spectral karyotyping with increased automation in chromosome analysis.

Comparative genome hybridization (CGH) (Kallioniemi et al. 1992) has proved a very powerful genome-wide approach in cytogenetics to detect gain or loss of chromosomal material, notably with clinical application in cancer genetics. Here the hybridization of test and reference samples of genomic DNA (labelled with different fluorochromes) to sets of normal human chromosomes can be compared. The use of microarray technology in combination with comparative genome hybridization (array CGH) (Box 4.2) (Pinkel et al. 1998) has dramatically improved our ability to define and quantify structural genomic variation, notably submicroscopic structural variation. This has lead to an appreciation of the extent of copy number variation among healthy individuals (discussed in detail in Section 4.2).

Human chromosomes first observed by Fleming and Arnold (1886)

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