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8.4: Cytogenetics –Introduction - Biology


The genetic material of humans is contained on 23 pairs of chromosomes in the nucleus of each somatic cell. Human females inherit 2 X sex chromosomes while males inherit 1 X and 1 Y chromosome.

Cytogeneticists examine chromosome structure, seek out causes of chromosomal abnormalities, and study how chromosome structure relates to an individual's phenotype (appearance and biochemical traits). Abnormalities in chromosomes can result in early embryonic death, congenital defects, development of cancer, and infertility or sterility.

Studies of chromosomes begin with the extraction of chromosomes from cells. Chromosomes are then placed on a glass slide, stained with dye, and examined under a microscope. Each chromosome pair is assigned a number (from 1 to 22, then X and Y) that is based on staining pattern and length. A karyotype is a representation of a person's chromosomes. The chromosomes are shown in pairs and arranged in order of decreasing size. Individual genes cannot be seen on karyotypes.

What to look for in a human karyotype

  • Are there 46 chromosomes?
  • Is there one pair of each autosome and 1 pair of sex chromosomes?
  • Are there any deletions, rearrangements, or other abnormalities in the chromosomes?

Chromosomal notation: Karyotypes are presented in a standard form. First, the total number of chromosomes is given, followed by a comma, and the sex chromosome constitution.

Normal human female is designated as 46, XX

Human male with an extra chromosome 15 is designated as 47, XY, 15+

Human female with an extra X chromosome is designated as 47, XXX

There are many disorders that can be diagnosed by examining chromosomes. In Down syndrome, an extra chromosome 21 is present. In Turner syndrome, a sex chromosome is missing so that the individual has 45 total chromosomes. Fragile X syndrome, the most common inherited cause of mental retardation, takes its name from the appearance of the X chromosome.


Cytogenetic Laboratory Management: Chromosomal, FISH and Microarray-Based Best Practices and Procedures

Cytogenetic Laboratory Management: Chromosomal, FISH and Microarray-Based Best Practices and Procedures is a practical guide that describes how to develop and implement best practice processes and procedures in the genetic laboratory setting. The text first describes good laboratory practices, including quality management, design control of tests and FDA guidelines for laboratory developed tests, and pre-clinical validation study designs. The second focus of the book describes best practices for staffing and training, including cost of testing, staffing requirements, process improvement using Six Sigma techniques, training and competency guidelines and complete training programs for cytogenetic and molecular genetic technologists. The third part of the text provides step-wise standard operating procedures for chromosomal, FISH and microarray-based tests, including pre-analytic, analytic and post-analytic steps in testing, and divided into categories by specimen type, and test-type.

All three sections of the book include example worksheets, procedures, and other illustrative examples that can be downloaded from the Wiley website to be used directly without having to develop prototypes in your laboratory.

Providing both a wealth of information on laboratory management and molecular and cytogenetic testing, Cytogenetic Laboratory Management will be an essential tool for laboratorians world-wide in the field of laboratory testing and genetics testing in particular.

This book gives the essentials of:

  • Developing and implementing good quality management programs in laboratories
  • Understanding design control of tests and pre-clinical validations studies and reports
  • FDA guidelines for laboratory developed tests
  • Use of reagents, instruments and equipment
  • Cost of testing assessment and process improvement using Six Sigma methodology
  • Staffing training and competency objectives
  • Complete training programs for molecular and cytogenetic technologists
  • Standard operating procedures for all components of chromosomal analysis, FISH and microarray testing of different specimen types

This volume is a companion to Cytogenetic Abnormalities: Chromosomal, FISH and Microarray-Based Clinical Reporting. The combined volumes give an expansive approach to performing, reporting and interpreting cytogenetic laboratory testing and the necessary management practices, staff and testing requirements.


Structure and Synthesis of Telomere

Molecular genetic studies have shown that the telomere consists of several short sequences which are tandemly repeated (Table 8.8). The general formula for the composition of the repeating units in one strand of the telomeres of all the eukaryotes is as follows:

where n is greater than 1 and m varies from 1 to 4. The other strand has its complementary sequence.

There exist single strand breaks in the telomeric region. The terminal part of the telomere is folded in some specific way so that neither ligase can seal it nor nucleases can degrade it. Some specific proteins are also bound to the telomeres and thereby afford additional protection.

Although one strand (G-rich strand) of the telomeric DNA is long and single stranded (unpaired), it forms a duplex structure through folding back upon itself. Two models have been suggested for the folding mechanisms at the telomeric ends.

1. Four G-rich repeating units (5′ TTGGGG3′) are involved in forming the duplex struc­ture. Two of the repeating units fold back on the other two to form a hairpin like structure within which G : G pairing occurs. (Fig. 8.9).

2. According to the alternative model, proposed by Williamson and coworkers in 1989, four repeating units (5′ TTGGGG 3′) fold in such a way that the second G of each unit acts as a member of a “quartet”. Rest of the nucleotides form a loop (Fig. 8.9). This model is known as G quartet model.

Synthesis of Telomeric DNA:

Synthesis of the telomeric repeating sequences has been well understood in Tetrahymena. In this species an enzymes called telomerase was discovered in 1987 by Greider and Blackburn this enzyme synthesize the telomeric DNA. The enzyme telomerase contains a short chain of single- stranded RNA made of 159 ribonucleotides thus the enzyme is a ribonucleoprotein.

The enzymatic RNA includes a 15-22 base long sequence which is similar to the C-rich sequence (3′ AACCCC 5′) (Table 8.8) and it is complementary to the G-rich sequence (5′ TTGGGG 3′) of the telomere. This RNA sequence functions as a template for the synthesis of the G-rich sequence (Fig. 8.10).

Thus the enzyme telomerase functions like “reverse transcriptase” During synthesis, the nucleotide G or T is added one-by-one at the 3′-OH end of the G-rich DNA primer. When the whole sequence is synthesized, a new cycle of DNA synthesis starts through the movement of the enzyme at the new 3′-OH end.

The length of the telomeric DNA increases by this process of synthesis. However, the telomeric length is controlled by some regulatory mechanism.

Yeast Artificial Chromosome (YAC):

A chromosome requires three necessary components for its existence:

(i) Telomeres to seal the ends of the chromosome and to ensure survival,

(ii) An origin of initiation of DNA replication and

(iii) A centromere for movement and segregation into daughter cells.

Yeast artificial chromosomes have been constructed by putting the above three elements together they can propagate in yeast. YACs are maintained in circular form before insertion of foreign DNA into them. The circular YAC has a BamHI cleavage site at the end of telomeric sequences, and Smal cleavage site at some other position.

In addition, it has a marker TRPX on one side of the centromere, while other marker URA3 is carried on the other side. The enzyme BamHI cleaves the circular YAC to produce a linear molecule whose both ends act as telomeres.

The other enzyme Smal cleaves this linear molecule into two pieces between which foreign DNA can be inserted (ligated) to produce a linear chromosome carrying different genes. The markers TRPX (in one arm) and URA3 (in the other arm) help in selection of the molecules in which the two arms have been joined. The DNA inserted may be 50-300 kbp in length, thus eukaryotic genes can be carried in YACs. The whole genomic library can be maintained in the YACs and can be propagated in yeast.