CYTOGENETIC

ABNORMALITITES INDICATIONS FOR CYTOGENETIC ANALYSIS

§ Prenatal – pregnancies involving older
CYTOGENETICS (>35yrs) women.
- the study of chromosomes and its
abnormalities: alteration in the number § Unexplained psychomotor retardation
and structure. with or without dysmorphic features.

MILESTONES IN CYTOGENETICS § Confirmation or exclusion of diagnosis -
ArnOld known chromosomal syndromes.
- First Observed chromosomes:1879
§ Abnormalities of sexual differentiation
Hansemann & Flemming and development - ambiguous genitalia.
- Counted the chromosomes: 1891
Continued…
Winiwarter § Recurrent miscarriage, stillbirth or
- Isolated X chromosome spontaneous abortions.
§ Females with proportionate short
Painter stature and primary amenorrhea
- Isolated Y chromosome (absence of menstruation).
§ Parents and children of persons with
Tijio and Levan: 1956 chromosomal translocations, deletions
- Described correct chromosome number and duplications.
as 22 pair of autosomes and 2 sex § Pregnancies at risk of aneuploidy
chromosomes. (presence of abnormal number of
chromosomes in a cell) from results of
Levan fetal ultrasound.
- introduced the use of Colchicine to § Neoplastic conditions- soft tissues and
arrest mitosis at metaphase. hematological.

Hsu,Makino & Nishimura and Hughes APPROACH TO THE DIAGNOSIS OF
- Hypotonic technique: In 1952 in CYTOGENETIC DISORDERS
karyotyping. 1. Fluorescence insitu hybridization
2. Spectral karyotyping
Gall and Prudue 3. Insitu hybridization
- described in situ hybridization 4. Comparative genomic hybridization
techniques. 5. Karyotyping

Chromosome
- Is a packaged
and organized
structure
containing the
DNA of a living
organism.
KARYOTYPE 2. CULTURE
- Standard display of stained and § Culture medium
photographed chromosomes in - Preservative free sodium heparin,
metaphase spread, arranged in pairs, in (RPMI 1640 ), mitotic stimulant
order of decreasing length. (phytohemagglutinin) and antibiotics(
penicillin, streptomycin ).
- Human somatic cells - 22 pairs of
autosomes identical in male and female § Short term culture
- 1-3 days – blood, bone marrow,
- 2 sex chromosomes chorionic villi
XX - female
XY - males § Long term culture
- 1-3 wks - other tissue types
1. TISSUE SAMPLES & CELL CULTURE
A. Prenatal STAINING
§ Chorionic villi Q-banding
- 25mg of Vascularized and budding villi - The first banding method developed
from chorion frondosum
- Uses quinacrine mustard or quinacrine
§ Amniotic fluid - 20ml dihydrochloride

§ PUBS - creates a fluorescent transfers band on
- Percutaneous Umbilical Blood exposure to UV light
Sampling
- Q- bands fade over time not routinely
B. Postnatal used.
§ Peripheral blood
- 4ml of heparinized
§ Skin fibroblasts
- 4mm diameter
§ Bone marrow
- 1 ml of heparinized bone marrow.
§ Lymph node
- 0.5 to 1 cm3
§ Solid tumors
- Part of specimen submitted for
histopathological examination. Ideally
0.5-1 gm






G-banding R-banding
- Uses Giemsa dye to produce - Treating chromosomes with a hot
transverse bands light (G-C rich DNA) alkaline solution BEFORE Giemsa
dark band (A-T rich DNA) staining

- G-bands are identical to Q-bands - Produces bands that are the reverse of
G-bands, called R- bands.
- G-banding is the most widely used
banding technique for routine - In R banding, TELOMERES should
chromosome analysis appear as DARK BANDS and their
absence as the result of deletion is more
- Around 400 bands per haploid genome obvious.
seen.

- Each band corresponds to
5-10megabases

SUCCESSFUL CYTOGENETIC ANALYIS
DEPENDS ON
- Cells must be in Adequate numbers
- Analysis must be performed on Viable
cells in division
- Chromosomes Must be separated from
one another
- Chromosomes Must be identified &
characterized normal/abnormal
- Arranged According to the length in a
decreasing order.

CYTOGENETIC ABNORMALITIES Eyes
1. Chromosomal disorders - Speckling of iris (Brushfield spots)
- Autosomes - Fine lens opacity,refractive error
- Sex chromosomes Nystagmus,strabismus,blocked tear duct
2. Cancer cytogenetic
- Soft tissue tumors Ears
- Hematological disorders - Small, overfolding of upper helix
- Small or absent ear lobes
DOWN SYNDROME - Hearing loss
- John Langdon Down in 1866
Skin
- Trisomy of chromosome 21 - Loose folds in posterior neck (infancy)
- Cutis marmorata – extremities
- 1 in 700 live births
Hands
- Major cause of mental retardation - Short metacarpal and phalanges
- Hypoplasia of mid phalanx of 5th finger
- Maternal age has a strong influence – as - Single palmar deep flexion crease-
the age increases the risk of down simian crease
syndrome increases







Feet
- Wide gap between 1st and 2nd toes
- rocker-bottom feet

General Cardiac
- Hypotonia with tendency to keep mouth - Endocardial cushion defects-40%
open - VSD,PDA,ASD,MVP
- Protruding tongue - AR by 20yrs of age
Craniofacial
- Brachycephaly with flat occiput
- Mild microcephaly
- Upslanting palpebral fissures
- Late closure of fontanelles
- Aplasia of frontal sinus
- Low nasal bridge
- Inner epicanthal folds

EDWARD SYNDROME PATAU SYNDROME
- TRISOMY 18 SYNDROME - TRISOMY 13 SYNDROME

- 1 per 6,000 newborn babies, <5% - 1 in every 5,000 births
survive to term
- 47,XY,+13
- 47, XY/XX+18

- Second most common autosomal
trisomy

TURNERS SYNDROME KLINEFELTERS (XXY) SYNDROME
- Henry Turner – 1938 - Harry Klinefelter – 1942

- Complete or partial monosomy of X - Male hypogonadism
chromosome characterized by
hypogonadism in phenotypic females - 2 or more X chromosomes and
1 or more Y chromosomes.
- 1 in 2000 live born females
- Most common cause of hypogonadism
- 45X SYNDROME - X0, and infertility

- 1 in 500 males affected

- Classic pattern
47XXY karyotype in 82% of cases

- Other mosaic patterns
46XY/47XXY
47XXY/48XXXY
48XXXY/49XXXXY

Karyotype 47,XXY


XXXXX SYNDROME / PENTA X SYNDROME CRI-DU-CHAT SYNDROME (5p-)
- First described by Dr.Nirmala kesaree - Deletion of short arm of chromosome 5
and Wooly in 1963. (5p-)
- Critical region:5p15
- Found the abnormality in prisoners in - 1 in 15,000 to 1 in 50,000
America.

 PRADER-WILLI SYNDROME ANGELMAN SYNDROME
- 1 in 15,000. - HAPPY PUPPET SYNDROME
Mechanism:
- Deletion of 15q at q11- - abnormal puppet like gait,
q13(paternally derived)- 75% characteristic facies, paroxysms of
laughter, due to brain stem defect-
- Maternal UPD – 2 maternal,no not apparently associated with
paternal copies of 15q - 20% happiness

- Chromosomal translocation Mechanism:
involving proximal 15q – 5% - Deletion of 15q11 (maternal origin)-
75%

- Paternal uniparental disomy in 2%

- Imprinting mutation 2%

*Happy disposition,
an open mouth expression, widely
spaced teeth, and a pronounced
mandible

CLINICAL APPLICATIONS OF CYTOGENETICS
IN HEMATOLOGICAL DISORDERS

1. Confirmation of the diagnosis of CML
2. Confirmation of blast crisis of CML
3. Diagnosis of Acute leukemias
4. Diagnosis of lymphoproliferative
disorders
5. Diagnosis of non hodgkins lymphomas