MT 6315 CYTOGENETICS- UNIT 1: INTRODUCTION TO CYTOGENETICS

LESSON 1: CYTOGENETICS DEVELOPMENT AND IMPORTANCE

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OVERVIEW OF CYTOGENETICS
o Provided a way to preferentially mark
1959: Individuals who did not nucleic acids and make them visible to
possess the normal sex the microscope
chromosomes were discovered o Provided some of the first images of
 Normal: XY – male ; XX chromosomes because he was able to
– female stain them with aniline
 Has led to the study of o Mostly used as a precursor for the
human CYTOGENETICS
chromosomes, synthesis of other organic compounds
 Branchrapid
of genetics
advancesthat studies the structure of DNA within 1888 Heinrich Wilhelm Gottfried von Waldeyer
the cell nucleus  Coined the term “chromosome” after staining
 Deals with the study of structures and properties of techniques had been developed
chromosomes o “Chromos” – color
o Which means colored body o “Soma” – body
o Behavior during somatic cell division during growth 1865 Gregor Mendel
and development  Father of Genetics
o Behavior during germ cell division during  Discovered paired factors
reproduction  Described patterns of inheritance through pairs
o Influence on phenotype of unit factors (later on known as genes) for
 Branch of science that deals with the study of karyotypes, each trait
metaphases, translocations, satellited, centromeres, and o Fundamental units of heredity
deletions, etc. o Travel unchanged from one generation
 deals with karyotyping, fluorescence in situ hybridization to the next
(FISH), chromosome structures, and aberration study  principle of pairing
 Focus on the study of human chromosomes and human o outward traits are driven by genes that
genomic structure, function, and variation and their role in exist in pairs or what we called as
human disease and heredity alleles
 Discovered through the study of garden peas
MOLECULAR CYTOGENETICS
(almost 30,000), “Pisum sativum”
 analysis of genomic alterations MENDEL’S LAW
 mainly uses in situ hybridization a. Paired factors are inherited
 clinical applications were promoted by the o From both parents
commercialization of several DNA probes b. Paired factors are segregated during
gamete formation
CYTOGENETICS DEVELOPMENT (Ferguson-Smith, 2015) c. Paired factors independently sort
o Do not influence each other when
 Before: use of conventional binding techniques sorting, to form a gamete
 Current: use of molecular array comparative genomic 1902 Theodore Boveri & Walter Sutton
hybridization  Developed the Chromosome theory of
o but conventional binding techniques are still being used inheritance
 With the combination of these conventional and molecular o Stated that Mendel’s genes are located
techniques, CYTOGENETICS has become an essential tool in a specific locus in the chromosomes
for the diagnosis of various genetic disorders, paving the o Supported the theory of Mendel
way for possible treatment and management (Kannan and
Alwi, 2009) DIFFERENCE WITH MENDEL’S THEORY
 Chromosomes are the one responsible
for inheritance (it is the genetic material),
instead of the paired factors indicated by
Mendel

A. CHROMOSOME (CELL) DISCOVERY

1840s Karl Wilhelm von Nageli CHROMOSOMAL THEORY OF
 Described thread-like structures in the nuclei of INHERITANCE
plant cell a. Chromosomes occur in pairs and are
 Termed these structures “transitory inherited from parents (paternal and
cytoblasts” (now known as chromosome) maternal chromosome)
b. Chromosomes segregate in gamete
formation (haploid)
1870 Walther Flemming o One set of chromosome (23)
 Described patterns introduced aniline staining o Each parent synthesizes gametes that
to observe chromosomes during cell division contains only half of their
chromosomal complement (equal
o Poisonous substance derived from coal
genetic contribution)
 c. Chromosome pairs segregate and X-ray diffraction studies of Rosalind
independently Franklin
1961 Crick, Brenner, Barnett & Watts-Tobin
FIRST GENETIC LINKAGE MAP  Discovery of the genetic code in protein
1881 Edouard-Gerard Balbiani synthesis
 Discovered “polytene chromosomes” in  Discovered codons
insects o Triplets of base pairs responsible in the
o Large chromosomes which have identity of the amino acid
thousands of DNA strands
o Provide a high level of function in
certain tissues such as salivary gland
- Thomas Hunt Morgan, Calvin Bridges, Alfred
Stultevart, & Herman Joseph Muller
 Constructed the first genetic linkage maps
 derived from recombination studies in crosses
made in fruit fly & cytological preparations of
its polytene salivary gland chromosomes
1920- Cyril Darlington
1930  Pioneered plant cytogenetics
o Genetic studies were mostly
confined to plants and animals
(before the emergence of human
C. HUMAN CYTOGENETICS EMERGENCE
cytogenetics)
1912 Von de Winiwarter
 made important advances in the understanding
 Proposed no. of chromosomes:
of mechanisms of “chiasma formation” and
o Testes: 47
behavior of sex chromosomes in meiosis
o Ovary: 48
CHIASMA or CHIASMATA  X sex chromosome
o Males: Single X
- the point of contact or the physical link between
o Females: Two X
two non-sister chromatids of homologous
1923 Theophilus Shickel Painter
chromosomes
- site where chromosomal crossing over happens  discovered the role of Y chromosome in the
o exchange of genetic material development of male embryo
Before IMPROVEMENTS IN CYTOGENETIC
1950 TECHNIQUES
- enabled discovery of the correct number of
chromosomes in 1956
 Colchicines: used to arrest cells in metaphase
o it was difficult to determine the
diploid number because
chromosomes are crowded in this
phase
 Hypotonic solution: used for better
chromosome visualization
1956 Joe Hin Tjio & Albert Levan
B. CHROMOSOME COMPONENTS  normal number of chromosomes was
AND STRUCTURE DISCOVERY discovered
1944 GENETIC TRANSFORMATION o through the use conventional
EXPERIMENT binding techniques
 DNA, and not proteins in chromosome, is the  such as staining of
hereditary material chromosomes using
o Responsible for hereditary in genes fluorochromes (examined
through the use of fluorescent
and chromosomes
microscope)
 This experiment made use of bacteria
 showed that the correct number of
1950 Erwin Chargaff
chromosomes is 46 in both sexes (23 pairs)
 Discovered the amount of nitrogen base in
o 23 from mother, 23 from father
chromosome
o Autosomes: 22 pairs
 Pyrimidines – single ring (C,T)
o Sex Chromosome: 23rd pair
 Purines – double ring structure (A,G)
1969 Capersson & Zech
CHARGAFF’S RULE
 introduced chromosome banding with
 Amount of adenine = thymine
quinacrine that intercalated into DNA
 Amount of cytosine = guanine
1972 Pardue & Gall
1953 James Watson & Francis Crick  Introduced in situ hybridization using
 double helix structure of DNA radioisotopes for mapping DNA sequences
 based their findings on the Chargaff’s rule into chromosomes
 o Labelling of radioisotopes is time
consuming, thus replaced by FISH
and UV microscopy
1980s Fluorescence in situ hybridization (FISH)
 became the standard method for gene
mapping
 this enabled detection of numerical
chromosome aberrations

FLUORESCENT IN SITU HYBRIDIZATION (FISH)

 developed late 1980s from radioactive hybridization

procedures
 initially developed for mapping human genes
 later on used in clinical cytogenetics laboratories for:
 Synaptinemal/Synaptonemal complex
- characterization of chromosomal rearrangements
o product of synapsis
- characterization of marker chromosomes
 close association of homologous
- detection of microdeletions
chromosomes)
- prenatal diagnosis of common aneuploidies
o Tripartite structure that holds the chromosomes
 has made cytogenetics into a molecular level
together
USES OF MODERN FISH TECHNIQUES  Neottiela rutilans – fungi usually used in chromosome studies
1. Visualization of deletion or rearrangement of a single because it has an ordinarily large nuclear DNA content
gene or chromosome translocation
2. Determination of the copy number of oncogenes COMPUTER INTERFACE IN USING FISH
amplified in tumor cells
3. Characterization of very complex rearrangements

INTERPHASE FISH APPROACH

 Genomic alterations can also be detected in all types of
human tissues
o w/out cell culture and chromosome preparation, like:
- touch preparations
- sections of frozen tumor
- paraffin-embedded tissue
SOME OF THE FISH TECHNIQUES:
1. Multiple color FISH (M-FISH)
2. FISH with multiple subtelomeric probes IMMUNOFLUORESCENCE MICROSCOPY

DETECTION OF NUMERICAL CHROMOSOMAL

ABERRATIONS/ ANEUPLOIDIES

 Trisomy 21 in Down syndrome

o Abnormality in the cell division in which full or partial
copy of Chromosome 21 occurred
 45, XO in Turner syndrome
o Missing one “X” in males (45 instead of 46)

 47, XXY in Klinefelter syndrome

o two or more X chromosomes in males

 Trisomy 13 in Patau syndrome

o Three copies of chromosome 13 instead of the normal
2 copies
 Trisomy 18 in Edward syndrome
o Three copies of chromosome 18

 Philadelphia chromosome in patient w/ Chronic

Myelogenous Leukemia (CML)
o Indicates good prognosis
 Antigen-antibody reaction
CHROMOSOME STUDIES o Use of antigens (antibody generator) for the body to
ELECTRON MICROGRAPH produce antibodies
o Antigen: ‘antibody generator’; foreign

IMPORTANCE OF CYTOGENETICS IN
HUMAN STUDIES
  molecular cytogenetics can detect chromosomal
 Cytogenetics is important in studying inherited and translocations in:
acquired anomalies o hematologic neoplasm
 The incidence of structural chromosome abnormalities o malignant lymphomas
which are visible at the level of 400 bands is approximately o solid tumors
1/200 at birth  detects involvement of specific genes

I. PRENATAL STUDIES LEUKEMIA

Chronic Myeloid  interphase FISH is highly
PRENATAL DIAGNOSIS OF THE COMMON Leukemia sensitive in detecting the
ANEUPLOIDIES BCR/ABL fusion
Mixed Lineage  involvement of the MLL
 Aneuploidy – abnormal number of chromosomes Leukemia gene in an 11q23
 Aneuploidies of chromosomes 13, 18, 21 rearrangement
o 95% of the chromosomal aberrations causing live- Childhood Acute  involvement of the
born defects Lymphoblastic TEL/AML1 fusion
 DNA process and FISH protocols were commercially Leukemia
standardized in 1990s
 Modern detection techniques LYMPHOMA
o Interphase FISH non-Hodgkin  fish probes for several rearranged
o Prenatal diagnosis with uncultured amniocytes Lymphomas genes are commercially available
Follicular  interphase FISH detection of the
cells cultured from amniotic fluid could be used to lymphoma BCL2 arrangement
determine the chromosome content of the fetus - using breakpoint-flanking
probes
 Early detection would provide early treatment or therapy - advantageous than standard
PCR method
NON-INVASIVE DETECTION OF CHROMOSOMAL
DISORDERS USING MATERNAL BLOOD ANALYSIS OF GAINS AND LOSSES OF CHROMOSOMES
OR CHROMOSOMAL REGIONS IN TUMORS
Cytogenetic analysis of fetal cells by FISH
Fetal nucleated RBCs pass into the maternal circulation  almost all types of clinical specimens can be used for
o provide a cell source for noninvasive prenatal comparative genomic hybridization (CGH) studies of
genetic diagnosis tumors
STUDIES OF MOSAICISM AND ITS EFFECTS ON EARLY  gains or losses is correlated with particular tumors and
HUMAN DEVELOPMENT different stages of the tumor
o can be used in the prognosis of patients
MOSAICISM
 presence of two different genotypes in an individual which
TESTING DELETION OF TUMOR SUPPRESSOR GENES
developed from a single fertilized egg
AND AMPLIFICATION OF ONCOGENES
 as a result, the individual will have two genetic cell lines
derived from a single zygote  detected by FISH or CGH studies of tumor tissues
 TUMOR SUPPRESSOR GENES: anti-oncogenes
TWO TYPES OF MOSAICISM o e.g. p53 and RB-1
1. MEIOTIC MOSAICISM  ONCOGENES: a gene which in certain circumstances can
- Occurrence of a mitotic error producing a diploid transform a cell into a tumor cell
cell line in a trisomic conception o N-myc for neuroblastoma
o C-myc and HER-2/neu for breast cancer
2. SOMATIC MOSAICISM (constitutional)
- trisomic cell line occurred in a conception which III. DETECTION OF SUBTELOMERIC
was initially diploid ABERRATIONS/ANOMALIES
CONSTITUTIONAL MOSAICISM
 Detection in patients with unexplained mental
 Result of post fertilization mitotic error, that is a
retardation or developmental disabilities
somatic event
 Genomic alteration in the subtelomeric regions can be
detected through cytogenetics
II. HUMAN CANCER STUDIES o associated to the cause of developmental
disabilities
DETECTION OF SPECIFIC CHROMOSOMAL o Second only to down syndrome as the most
TRANSLOCATIONS AND GENE REARRANGEMENTS common cause of mental retardations
 studied by FISH w/ multiple subtelomeric probes
 Qualitative aberration
o The abnormality is not in the number of chromosomes,
but rather on how it appears IV. MICRODELETION SYNDROMES DETECTION
 use to determine a therapy plan, monitoring treatment,
MICRODELETION
and predicting prognosis
  a syndrome caused by chromosomal deletion smaller
than 5 million base pairs spanning several genes
 very small for high resolution karyotyping or
conventional cytogenetic methods to detect

MICRODELETION SYNDROMES
1. Williams
2. Prader-Willi/Angelman
3. Smith-Magenis
4. 22q11.2 deletion
 Associated with a number of syndromes,
o e.g. conotruncal anomaly face syndromes
 Also associated with psychiatric illnesses,
o e.g. schizophrenia
5. 1p36 deletion
 most common microdeletion syndrome
ARM OF A CHROMOSOME
 p – short arm, “petit”
 q – long arm