Cytogenetics Typical ‘chromosome spread’ Chromosomes obtained from actively-dividing mitotic cells Jo Sanders Dept of Haematology, Christchurch Hospital, New Zealand Cytogenetics • Study of chromosomes and their abnormalities • Chromosomes are genetic structures of cells containing DNA • Each chromosome has a characteristic length and banding pattern • All cells contain 23 pairs of chromosomes (46 in total), and each chromosome contains thousands of genes • Specimens: Peripheral Blood, Bone Marrow, Tumour, Solid tissue • The chromosomes are spread out, fixed and stained to allow examination down the microscope • Abnormalities are identified by changes in banding patterns along the chromosome. • A karyotype is made which shows the chromosomes of an individual arranged in pairs and sorted according to size 1 Chromosome Labeling Chromosome is Each arm Each sub-region identified with a divided into divided into number ranging sub-regions bands identified 1-22,or X and Y and identified with a number by a number Example - • The first chromosome, long arm, second region of the chromosome, the fourth band of that sub-region A Karyotype • Light microscope used to view chromosomes in metaphase of mitosis • Chromosomes arranged into homologous pairs based on size and banding patterns 1 2 3 4 5 • The normal human karyotype is made of 46 chromosomes: • 22 pairs of autosomes, 6 7 8 9 10 11 12 numbered from 1 to 22 by order of decreasing length • 1 pair of gonosomes, or sex 13 14 15 16 17 18 chromosomes: XX in the female, XY in the male. 19 20 21 22 X Y Importance of Karyotypes • Karyotypes show the chromosomal makeup of an individual. Knowing the number of chromosomes is essential for identifying chromosomal variations that cause genetic disorders or idenifiying malignancies ie. APML, CML 2 Chromosome nomenclature • Patterns, and the nomenclature for defining positional mapping have been standardised to allow cytogeneticists to communicate and archive information for medical purposes • Numbering begins from the centromere and continues outward to the end of each arm. Conventionally, the arms are divided into a number of regions by means of easily recognisable "land-mark" bands, and bands numbered sequentially within each. Sub-bands are catered for by using a decimal system Chromosome structure short arm = p (petite) Centomere Chromatid long arm = q Telomere Anomalies • Chromosomes are genetic material and therefore carry the organisation of cell life and inherited traits • Cell life will be disturbed if regular segregation fails this can occur during embryogenesis (constitutional anomalies) or in cancer (acquired anomalies). 3 Constitutional anomalies • All the tissues ("the whole patient") hold the same anomaly. The chromosome error was already present in the embryo. • "Constitutional anomalies" thus refer to the chromosome inborn syndromes, such as trisomy 21, Turner syndromes, and others. Acquired anomalies • Only one organ is involved, the other tissues of the body are normal. • The patient has a cancer of the affected organ. "Acquired anomalies" thus refer to malignancies. Mosaic anomalies • When only some cells carry the anomaly whilst others are normal ( or carry another anomaly) • Very common in leukaemias and other cancers subject to continuous chromosome change • In ALL there may be a normal clone, one clone with a specific change, and a third with additional changes (46, XY / 46, XY, t(4;11) / 46, XY, t(4;11), I(7q)) ALL (46, XY / 46, XY, t(4;11) / 46, XY, t(4;11), I(7q)) t(4;11), I(7q)) 4 Variations in Chromosomal Number • Euploidy – the normal number and sets of chromosomes • Polyploidy – the presence of three or more complete sets of chromosomes • Aneuploidy – the presence of additional or missing individual chromosomes Types of Polyploidy • Triploidy – three sets of chromosomes 23 x 3 = 69 • Tetraploidy – four sets of chromosomes 23 x 4 = 92 Types of Aneuploidy • Monosomy – one less chromosome (23 x 2) – 1 = 45 • Trisomy – one additional chromosome (23 x 2) + 1 = 47 Structural anomalies • Structural changes occur within the chromosomes but may not necessarily be accompanied by any numerical change. – if there is not loss or gain of genetic material the change is balanced – if there is deletion and/or duplication of chromosome segment(s) the change is unbalanced 5 AML Philadelphia Chromosome (Ph) • CML is an acquired cytogenetic abnormality that is characterised by the presence of the Philadelphia Chromosome (Ph) • The Ph chromosome is a result of an exchange of material (translocation) between the long arms of chromosomes 9 and 22 eg t(9;22) • This translocation brings together the BCR gene on chromosome 22 and the ABL gene on chromosome 9 • The resulting hybrid gene BCR/ABL causes uncontrolled cell growth The t(9;22) translocation 6 FISH for BCR/ABL • Fluorescent in situ hybridization (FISH) is a molecular cytogenetics technique that uses a fluorescent-labeled DNA probe to determine the presence or absence of a particular segment of DNA — the BCR-ABL gene in the case of CML • FISH can detect one leukemic cell in 500 normal cells CML Deletion • Loss of a segment of chromosome • Invariably, but not always, results in the loss of important genetic material – and is sometimes known as ‘partial monosomy’ • Recorded as del, followed by a bracket with the number of the chromosome, and a second bracket indicating the breakpoint(s) and the deleted region (eg del(5)(q14q34)) 7 Reciprocal translocation • A mutual exchange between terminal segments from the arms of 2 chromosomes • Recorded as t, followed by a bracket with the numerals of the 2 chromosomes, and a second bracket indicating the presumptive breakpoints eg AML t(15:17) Inversion • Inversion occurs when a segment of chromosome breaks, and rejoins within the chromosome effectively inverts it • Recorded as inv, followed by a bracket with the number of the chromosome, and a second bracket indicating the breakpoints, where these can be determined (eg inv(9) (p11q13)) Isochromosome • Loss of a complete arm, “replaced” by the duplication of the other arm (equivalent to a monosomy for one arm and a trisomy for the other). • Recorded as i, followed by a bracket with the number of the chromosome and the arm (eg i(17q) or i(17)(q10): duplication of the q arm and loss of the p arm) 8 Insertion • A segment of chromosome is deleted and transferred to a new position in another chromosome, or rarely within the same chromosome. • Recorded as ins, followed by a bracket with the number of the chromosome which receives the segment preceding the number of the chromosome which donates the segment eg ins(2)(p13q31q34) and ins(5;2)(p12;q31q34): the segment q31q34 from a chromosome 2 is inserted respectively in p13 of this chromosome 2, and in p12 of a chromosome 5. Duplication • Direct: a segment of chromosome is repeated, once or several times, the duplicated segment keeping the same orientation with respect to the centromere • Inverted: the duplicated segment takes the opposite direction • Recorded as dup, followed by a bracket with the nos of the chromosome, and a second bracket indicating the breakpoint(s) and the duplicated region If in doubt ask • Cytogenetics lab • Haematologist / Oncologist • CIMBTR 9.