the basics of the cytogenetics techniques.ppt
- 2. • What is Cytogenetics? Study of chromosomes; for clinical cytogenetics, it is applying the study of chromosomes to clinical medicine (diagnosis - postnatal, prenatal, cancer; gene mapping) • What types of tissues can be used to study chromosomes? for routine chromosome analysis, cells must have a nucleus, be viable at time of collection and capable of undergoing cell division (cells are grown in culture).
- 3. Why perform clinical cytogenetic testing? • Fertility problems – couples with history of infertility or Habitual pregnancy loss; females with amenorrhea. • Malformed fetus; stillbirths and neonatal deaths where a cytogenetic basis is likely. • Pregnancy in women of advanced age. • Family history in first degree relatives. • Dysmorphology, problems of early growth and development. • Neoplasia (can be diagnostic or prognostic).
- 4. Frequency of Chromosome Abnormalities • ½ to 1% of all live births. • 2% of recognized pregnancies in women > 35 y.o. • 10% of stillbirths. • ½ of recognized spontaneous pregnancy losses • High in neoplasias (type dependent)
- 5. Microscopically, each chromosome has two arms: i. Labeled p (the shorter of the two) is named for petit meaning small. ii. q (the longer). the q arm is named q simply because it follows p in the alphabet. (According to the NCBI, "q" refers to the French word "queue" meaning 'tail'.) Chromosome structure
- 7. Nucleosome • Winding of DNA on to histones is the first step. • Histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes.
- 8. • There are five histones: H1, H2a, H2b, H3, and H4. • These proteins' basic amino acids bind to the acidic phosphate groups on DNA.
- 9. Interphase chromatin During interphase (the period of the cell cycle where the cell is not dividing), two types of chromatin can be distinguished: Euchromatin: which consists of DNA that is active, e.g., being expressed as protein. Heterochromatin: which consists of mostly inactive DNA. It seems to serve structural purposes during the chromosomal stages.
- 10. Heterochromatin can be further distinguished into two types: • Constitutive heterochromatin: which is never expressed. It is located around the centromere and usually contains repetitive sequence. • Facultative heterochromatin: which is sometimes expressed. • Both play a role in the expression of genes.
- 11. A centromere is a region of DNA typically found near the middle of a chromosome where two identical sister chromatids come closest in contact. At the nucleotide level, the centromere is composed of a set of highly repetitive alpha satellite sequences, The mammalian centromere is composed of highly repetitive α-satellite DNA that consists of 171 bp monomer sequences.
- 12. Centromeres can be metacentric, sub metacentric, acrocentric or telocentric manner
- 13. karyotype karyotype is “a visual representation of an individual’s chromosomes arranged in a specific way. ”Karyotyping chromosomes involves classifying and organizing them according to the arrangement, number, size, shape, or other characteristics of the chromosomes. Typical Female Karyotype Typical Male Karyotype
- 15. • Polyploidy: •Complete extra set of chromosomes –Three of every chromosome –Cannot survive to birth. • Aneuploidy: •Missing or extra of one chromosome –Monosomy –missing one chromosome –Trisomy –one extra chromosome –Only Trisomy 13, 18 and 21 are viable Abnormal Number:
- 16. (Autosomal aneuploidy) Trisomy 21 Trisomy 21 is the presence of 3 chromosome 21. Trisomy 21 causes the condition commonly known as Down’s syndrome. The extra chromosome leads to the specific characteristics of Down syndrome, some of which are very familiar. However, not all individuals with Down syndrome will show the exact same characteristics there is a great deal of variability
- 17. (Autosomal aneuploidy) Trisomy 13 Trisomy 13 is the presence of 3 chromosome 13 Trisomy 13 causes the condition sometimes known as Patau syndrome. Trisomy 13 is a very serious condition. Only about 5% of babies with the disorder survive past their first year. Most pregnancies involving Trisomy 13 end in miscarriage. Children with Trisomy 13 usually have a lot of trouble breathing, especially when they sleep, and many have seizures. All individuals with Trisomy 13 have severe mental retardation.
- 18. (Autosomal aneuploidy) Trisomy 18 Trisomy 18 is the presence of 3 chromosome 18 Trisomy 18 causes the condition sometimes known as Edwards syndrome. Trisomy 18 is another very serious condition only about 10% of babies with the disorder survive past their first year. Most pregnancies involving Trisomy 18 end in miscarriage. Children with Trisomy 18 usually have breathing problems, difficulty eating, and many have seizures. Some have serious heart conditions. All individuals with Trisomy 18 have severe mental retardation.
- 19. (Sex chromosome aneuploidy) 47, XXY. An individual with the genotype 47, XXY is male. The extra X chromosome leads to features of the condition commonly known as Klinefelter syndrome. It affects about 1 in 1000 males. Most males are taller than average and may have a different distribution of body fat (e.g. more than usual in the hips or chest). Also tend to have sparse facial and body hair. Some have a degree of mental retardation, but many have normal intelligence. The most common feature of Klinefelter syndrome is infertility.
- 20. (Sex chromosome aneuploidy) Monosomy (Turner syndrome 45, X0) An individual with the genotype 45, X0 is phenotypically female. The person has 45 chromosomes instead of the usual 46. Instead of a trisomy, this would be called a monosomy. Monosomy X is the only monosomy known to be compatible with life. Having only one copy of an X chromosome leads to the features of the condition known as Turner syndrome.
- 21. Turner syndrome Affects about 1 in 5000 newborn females. Females with Turner syndrome are shorter than average and have other noticeable physical features. Swelling of the hands and feet, webbing of the neck, broad chest. May also have features which affect their health.
- 22. SEX CHROMOSOMAL ANEUPLOIDIES: TRIPLE X SYNDROME Triple X syndrome, also known as trisomy X and 47,XXX, is characterized by the presence of an extra X chromosome in each cell of a female. Those affected are often taller than average. Usually there are no other physical differences and normal fertility. Occasionally there are learning difficulties, decreased muscle tone, seizures, or kidney problems.
- 23. SEX CHROMOSOMAL ANEUPLOIDIES:YY SYNDROME XYY syndrome, also known as YY syndrome or Jacobs syndrome, is a genetic condition in which a human male has an extra male (Y) chromosome giving a total of 47 chromosomes instead of the more usual 46. This produces a 47,XYY karyotype which occurs every 1 in 1,000 male births. The clinical phenotype is normal and the vast majority of XYY males do not know their karyotype.
- 24. Chromosomal deletion syndromes result from deletion of parts of chromosomes. Depending on the location, size, and whom the deletion is inherited from, there are a few known different variations of chromosome deletions. Chromosomal deletion syndromes typically involve larger deletions that are visible using karyotyping techniques. Smaller deletions result in Microdeletion syndrome, which are detected using fluorescence in situ hybridization (FISH). Examples of chromosomal deletion syndromes include 5p-Deletion (cri du chat syndrome). Deletion
- 25. Translocation Non-homologous chromosomes have exchanged pieces (crossed over) 1.Robertsonian Translocation –Two q arms of two different chromosomes come together –Two p arms are lost entirely 2.Reciprocal Translocation –Two different chromosomes exchange parts –Since all parts are still present –often normal
- 26. Abnormal Structure • Isochromosomes: -Have two identical arms -Two p’s or two q’s and not the other
- 27. RING CHROMOSOME -Telomeres are lost, or don’t function -So one end of chromosome attaches to other end forming a ring -Cannot undergo mitosis successfully -Ring chromosomes may form in cells following genetic damage by mutagens like radiation.
- 28. INVERSION An inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end to end. An inversion occurs when a single chromosome undergoes breakage and rearrangement within itself. Inversions are of two types: paracentric (centromere is not involved)and pericentric (centromere is involved).
- 29. FRAGILE X SYNDROME most frequent inherited mental retardation • symptoms: Big head, elongated face, big ears, mild – severe mental retardation. Xq27.3 –increased fragility
- 30. • Fragile X syndrome occurs due to a mutation in the FMR-1 gene (Xq28) (FMR-1 protein is a RNA-binding protein), which leads to CGG trinucleotid-repeat expansion, followed by methylation and inhibited expression.
- 31. How Scientists Read Chromosomes? 1) Size: (large, medium, small) . 3) Centromere position : metacentric , submetacentric , acrocentric . 4) Banding pattern: The size and location of bands on chromosomes make each chromosome pair unique. To "read" a set of human chromosomes, scientists first use three key features to identify their similarities and differences:
- 32. Chromosome Landmark • Chromosomes appear as a continuous series of light and dark areas, called bands depending on their nucleotide and protein composition. • The band width and the order of bands is characteristic of a particular chromosome. • Each chromosome (1,2,3...22, X and Y) displays a unique banding pattern, analogous to a "bar code", which allows it to be easly differentiated from other chromosomes of the same size and centromeric position.
- 33. Chromosome Landmark • Chromosome region area lying between adjacent landmarks, ranges 1-4, numbered sequentially from centromere to telomere • Regions are divided into bands, numbered sequentially from centromere to telomere. (1q31).(21q22).(19p13)
- 34. Chromosome Landmark • Chromosome bands: part of a chromosome that is distinguished from adjacent parts by appearing darker or lighter with one or more banding techniques • In high resolution banding, bands are divided into sub- bands. (1q31.1).
- 36. Chromosomes are divided into 7 groups, A………G • Group A: 1,2,3 • Group B: 4,5 • Group C: 6-12, X • Group D: 13,14,15 • Group E: 16,17,18 • Group F: 19,20 • Group G: 21,22,Y
- 37. Types of banding • G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. The dark regions tend to be heterochromatic and GC rich. The light regions tend to be euchromatic and GC poor. • R-banding is the reverse of G-banding (the R stands for "reverse"). C-banding: Giemsa binds to constitutive heterochromatin, so it stains centromeres. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in G- banding. T-banding: visualize telomeres.
- 39. Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Spectral karyotyping
- 40. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) uses fluorescent molecules to “paint” genes or chromosomes. This technique is for gene mapping, identification of chromosomal abnormalities and identification of cultured/uncultured microorganisms in environment. FISH involves the use of short sequences of single-stranded DNA (probes) which are labeled with fluorescent tags, to hybridize, or bind, to the complementary DNA to see the location of those sequences of DNA under the fluorescent microscope.
- 41. Requirement for FISH There are 3 main component for FISH 1- sample 2- fluorescent probe 3- fluorescent microscope