Cytogenetics

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Cytogenetics
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Historical Gleanings

• The concept of heredity dates back to 6000yrs
• Haemophilia was 1st heredity disorder known (1500
  yrs).
• Aristotle suggested semen originated from blood
  in 3rd century.
• In 17th cen Dutch scientist Graaf demonstrated
  union of sperm ovum.

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Historical Gleanings

• Gregor Mendel
• Scientific approach to genetics came in 19th
  century with his discovery of principles of
  heredity.
• He showed that transmission of characters follow
  statistical laws.
• 3 laws
• Law of uniformity.
• Law of segregation.
• Law of independent assortment.

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Genetics

• Study of genes of statistical laws that govern
  passage of genes.
• 1902 William Bateson coined term Genetics.
• 1909 term Gene was coined by Johannesen.
• Genes are fundamental units of heredity.

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Branches of Genetics

• Cytogenetics.
• Molecular genetics
• Biochemical genetics
• Cancer genetics
• Immuno genetics
• Developmental genetics
• Behavioural genetics
• Population genetics

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Importance of genetics

• Chromosomal abnormality contribute to 1st
  trimester abortion.
• Congenital malformation of genetic origin in 2-3
  newborn.
• gt ½ childhood blindness, deafness, mental
  retardation is cos of genetic disorder
• gt5 of adult suffer from genetic disorder
• Many cancers have genetic component

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CELL DIVISION

• Multiplication of cells takes place by division
  of pre-existing cells
• This is an essential feature for development of
  embryo
• Cell divides by 2 ways
• Mitosis
• Meiosis

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• MITOSIS
• Daughter cells have genetic material
  chromosomal number similar to the mother cell
• Takes place in 4 stages
• Prophase
• Metaphase
• Anaphase
• Telophase

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• MEIOSIS
• It occurs only in gametes
• Two divisions viz. 1st 2nd meiotic divisions
• Here, the cells produced differ from other cells
  in that
• The number of chromosomes is reduced to half the
  normal number
• The genetic information is not identical in the
  various gametes produced

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Cytogenetics

• Its study of chromosomes cell division
• The term chromosome derived from greek
  chroma-color soma-body
• They are present on nucleus
• Interphase chromosome has coiled extended
  portion.
• Coiled portion appears dark-heterochromatin
  extended portion pale staining -euchromatin

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• In metaphase consist of 2 chromatids joined at
  centromere
• Chief chemical constituent is DNA
• Small units of heredity genes located on specific
  portion of chromosome called Locus.

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• There r 46 chromosome in somatic cell
• 44 autosomes 2 sex chromosomes
• 44 autosomes consist of 22 pairs. 1 chromosome of
  each pair comes from father mother.
• Sex chromosomes are X Y.
• Female has 44 XX. Male has 44 XY

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• Average size of metaphase chromosome is around
  5mm
• Shape interphase - thin thread like
• metaphase - thick rod like
• anaphase - rod or v or j shaped

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structure
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Classification of chromosome
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s
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• Standard classification (Denver classification)
• A to G.
• A-1 2 3 B 4 5 C- 6 7 8 9 10 11 12 X.
• D- 13 14 15 E- 16 17 18
• F-19 20 G-21 22 Y.
• This classification is based on Descending length
  of chromosome.

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Paris nomenclature

• Chromosomes are identified based on banding
  technique
• Proposed in Paris in 1971
• In this long short arm divided into regions
  1,2,3 n regions are subdivided to bands.
• Ex-RB gene -13q14 I,e long arm of chromosome
  13,4th band on 1st region

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KARYOTYPING

• Chromosomal constitution of individual is
  karyotype
• Karyotyping is a standard arrangement of a
  photographed or imaged stained metaphase spread
  in which chromosome pairs are arranged in order
  of decreasing length

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• SAMPLE PROCUREMENT
• To produce a karyotype, one must obtain cells
  which are capable of growth division
• Collection of 5 ml of venous blood
• mixed with heparin to avoid clotting
• Lymphocytes are separated from the red cells
• White cell suspension is put in culture vial
  which contains culture media, fetal calf serum
  phytohaemagglutinin
• Incubated for 3 days at 37C

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• After obtaining the sample
• Addition of colchicine
• Arrest of cellular division in metaphase by
  inhibition of microtubule formation, thus
  obstructing the completion of mitotic cycle
• Exposure to hypotonic solution
• Swelling of the cell for enhanced spreading of
  the chromosomes
• 31methanol/glacial acetic acid mixture
• Fixation of cells

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• Staining (banding)
• Microscopic analysis photography
• Karyotype production
• interpretation

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Chromosome Banding

• G Banding
• Most commonly used method
• Chromosomes are first treated with trypsin. It
  denatures chromosome protein
• Then, stained with Giemsa solution that stains
  each chromosome showing a unique pattern of light
  dark bands
• Q Banding
• Chromosomes are stained with quinacrine mustard

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• R Banding
• If chromosomes are pre heated before staining
  with Giemsa, then this gives a banding pattern
  that is reverse of G banding
• C Banding
• Here, the centromeric the regions of secondary
  constriction are stained
• Pretreated with acid than alkali stained wit
  giemsas

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R
G
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Q banding
C banding
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• High Resolution Banding
• In this, cells are arrested before metaphase (i.e
  in prophase or pro-metaphase)
• This provides greater sensitivity to see more
  number of bands compared to G banding
• NOR staining
• Here ammoniacal silver stain is used to stain
  nucleolar organizing regions

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NOR banding
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Karyotype Analysis

• Normally described using a short hand system of
  notations
• Total number of chromosomes
• Sex chromosomes complement
• Describe any abnormality
• Ex
• Downs syndrome 47 XY 21
• Turner syndrome 45 X0

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• If structural abnormality is present, mention
  whether on long arm or short arm
• Banded Karyotype
• Each arm of chromosome is divided into 2 or more
  regions by prominent bands
• Regions are numbered 1, 2, 3 from the centromere
• Each regions is further sub-divided into bands
  sub bands are ordered numerically
• Notation when there is structural abnormality
  xp21.2

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Classification of chromosomal anomalies

• Numerical (usually due to de novo error in
  meiosis) Aneuploidy - monosomy -
  trisomy Polyploidy - ttriploidy
• Structural (may be due to de novo error in
  meiosis or inherited) Translocations -
  reciprocal - Robertsonian (centric
  fusion) Deletions Duplications Inversions
• Different cell lines (occurs post-zygotically)
  Mosaicism

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Disorders number

• Monosomy
• cell with missing chromosome i.e. 45
• Ex TURNER SYNDROME
• Trisomy
• cell with 3 copies of chromosome i.e. 47
• Ex DOWNS SYNDROME
• Tetrasomy
• if individual has 4 copies of same chromosomes

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• Another type of numerical abnormality is
  polyploidy
• Complete set of chromosome has 22 autosomes 1
  sex chromosome, which is haploid
• If cells have
• 3 haploid chromosome triploid (69)
• 4 haploid chromosome tetraploid (92)

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Downs syndrome or Mongolism

• Also known as Trisomy 21
• 1 in 700 new born
• Described by Dr Langdon Down in 1866
• Chromosomal basis explained by lejeune in 1959
• Most Common Cause
• Meiotic non disjunction
• Others
• Translocation (Robertsonian) - 3
• Mosaicism

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• Risk according to maternal age-
• 1/1500 live birth for women of 20 yr age
• 1/30 for women 45 yr old

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• Complications
• Cardiovascular-ASD, VSD, Ostium Primum
• AV Malformations
• 10-20 fold increased risk for ACUTE LEUKEMIAS
• Both ALL AML
• Patients of 40 yrs develop
• Alzheimer disease
• Poor immunity
• Thyroid autoimmunity

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• TRISOMY 13 (PATAU SYNDROME)
• 1 in 4500 births
• First observed by Patau
• 95 babies die in a month
• If they survive - severe physical mental
  retardation seen
• Associated with increased maternal age

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• dia

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CLINICAL FEATURES

• Micro-ophthalmia, microcephaly mental
  retardation, cleftlip, cleft palate.
• Cardiac defect.
• Renal defect.
• Umblical hernia.
• Polydactaly.
• Rocker bottom feet .
• Extra finger or malformed thumb

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TRISOMY 18 (EDWARDS SYNDROME)

• Discovered by Edward in 1960
• 1 in 6000 live births
• 95 abort, 5 survive to term
• Many infants die within a month
• If they survive life span is 15 years

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CLINICAL FEATURES

• Retarded growth and development
• Hypertonia
• Prominent large head
• Low set malformed ears
• Small chin, small sternum
• Clenched fists(due to abnormal insertion of
  tendons)
• Rocker bottom feet

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• OTHERS
• VSD
• Omphalocele, diaphragmatic hernia
• Spina bifida
• High Mortality due to
• aspiration pneumonia
• Apnoea
• CHD
• Infections

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Numerical abnormalities of sex chromosomes

• Trisomies
• XXY
• XYY
• XXX
• Monosomy
• 45XO
• Mosaicism
• 46XY/46XX

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TURNER SYNDROME

• Also known as X monosomy
• Described by Turner in 1938
• Cytogenetic abnormality was shown by Ford in 1959
• 20 abort spontaneously
• 1 in 5000 to1 in 10000

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• GENOTYPE
• Monosomy 45XO
• Mosaicism 45XO/46XX
• Isochromosome 46 X I(Xq)
• Ring chromosome 46,X,r(X)

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CLINICAL FEATURES

• Short stature
• Low set hair line
• Webbing of neck
• Lack arithmetical skills
• Broad chest and widely spaced nipples
• Cubites valgus

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Webbing of neck
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• CVS
• Coarctation of aorta
• VSD
• Bicuspid aortic valve
• Urinary tract
• horse shoe kidney
• renal hypoplasia
• duplication of ureter

• Genital system
• streak ovaries
• small uterus
• primary amenorrhoea

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KLINEFELTER SYNDROME

• Genotype - 47 XXY
• 1st described by Harry Klinefelter in 1942
• Karyotype demonstrated by Jacobs Strong in 1959
• Individual has male phenotype with X chromatin
  ve
• 1 in 600 new born males
• Most Common Cause of hypogonadism in male

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CLINICAL FEATURES

• Difficult to diagnose till puberty
• Affected male is tall, thin, mental retarded
• Very small testis. Scrotum penis show
  hypoplasia
• Gynecomastia, wide hip in some cases
• Pubic, chin, chest, axillary hair absent or
  poorly developed

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• Testicular biopsy
• hyalinization of seminiferous tubules
• Spermatogenesis absent - patient is sterile
• Hormonal Profile
• Low testosterone level
• Pt are at high risk for
• Breast Cancer
• Germ Cell Tumor
• SLE
• Osteoporosis

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• Karyotype - usually 47 XXY
• 15 - mosaicism found i.e. 46XY/47XXY
• 60 - extra X chromosome is from
  meiotic/postzygotic non disjunction of maternal X
  chromosome 47XmXmY
• 40 - non disjunction of X X chromosome occurs
  during spermatogenesis 47 XmXpY

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47XYY

• 1 in 1000 newborn
• It results from non disjunction during 2nd
  meiotic division producing YY sperm
• Normal tall, male, slight mental retardation show
  emotional immaturity impulsive behaviour

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47XXX

• 0.1 in females
• Show 2 barr bodies
• Normal females with normal IQ reproductive life
• Females with more than 3x chromosome are severely
  mentally retarded sterile

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STRUCTURAL ABNORMALITY

• It results because of chromosomal breakage
• It may be spontaneous
• Other cause X-rays, chemicals, viral infections
• Important structural abnormality
• Deletion
• Inversion
• Ring chromosome
• Isochromosome
• Translocation

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• Deletion In this kind of structural
  abnormality, breakage occurs in part of
  chromosome broken part is subsequently lost as
  it has no centromere
• 2 types microscopic sub-microscopic
• Microscopic-terminal, interstitial

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• TERMINAL DELETION
• Examples
• Deletion of short arm of chromosome 5
• Its known as cri-du-chat syndrome
• Deletion of short arm of chromosome 4
• Its known as Wolf - Hirschhorn syndrome

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• Cri du chat
• 1 in 50000 births
• 1st described by Lejune his associates
• Cry of Affected newborn mimics meowing of cat
• Microcephaly, hypertelorism, low set ear,
• Severe mental retardation

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• Submicroscopic microdeletion

SYNDROME CHROMOSOME INVOLVED
Prader-willi 15
Angelman 15
Wilms tumour 11
Di - George 22
Miller Dieker 17
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Translocation

• Here exchange of genetic material between 2
  chromosome takes place
• May be balanced or unbalanced
• 2 types
• Reciprocal
• Robertsonian

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• 1 in 500
• Occurs between 2 non-homologous chromosome
• Exchange of chromosomal material distal to break
• No detectable phenotypic effect in carrier
• Mc- between chromosome 11 22

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• Robertsonian translocation
• 1 in 1000
• This results due to break at or near centromere
  in 2 acrocentric chromosomes subsequent fusion
  of their long arms

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INVERSION

• Involves single chromosome which breaks at 2
  point
• Broken segment rearranges itself by inverting its
  position
• 2 types pericentric paracentric
• In pericentric both arms(p,q) involved
• In paracentric either of arms involved

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Ring chromosome

• Its rare abnormality
• It involves 2 break at terminal region followed
  by fusion of cut ends
• Found in 1/5th cases of Turner syndrome

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Isochromosome

• Its due to abnormal splitting of centromere

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Mosaicism

• Its a condition in which individual has 2 or
  more cell lines
• 2 different cell lines are derived from single
  zygote
• Ex male mosaic - 46xy/47xy

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Chimera

• Is an individual has 2 or more genetically
  distinct cell lines derived from more than 2
  zygote

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• Chromosomal abnormalities can present in many
  ways
• Every child with Congenital anomalies should be
  evaluated by chromosomal studies.
• It prevents further unpleasant investigations
  being undertaken.
• To give information on prognosis.
• To help with details of relevant support groups.
• To know risk of recurrence for further siblings.

• Indications
• Multiple congenital anomalies.
• Unexpected mental retardation.
• Recurrent miscarriage.
• Unexplained stillbirth
• Sexual ambiguity or abnormality in sexual
  development.
• Infertility.
• Malignancy and chromosome breakage syndromes

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references

• Human embryology 7th edition I.B.Singh
• Human Genetics 3rd edition S.D. Gangane
• Medical genetics G.P.Pal
• Emerys Elements of medical genetics.
• Robins 8th editions