1. Cytogenetic Principles

2. Clinical Cytogenetics: The study of abnormalities of chromosome number and structure in relation to human disease
Cytogenetic abnormalities
collectively more common than all Mendelian single gene disorders together
Account for about 1/154 live births (0.65%)
Incidence in mothers > 35 years of age increases to about 1/50
Indications for Cytogenetic Analysis
Problems with early childhood growth and development
failure to thrive, abnormal physical appearance and/or internal structural abnormalities, mental retardation, ambiguous genitalia
Stillbirths and neonatal deaths that have the appearance of a cytogenetic abnormality
History of infertility or recurrent miscarriage
Known or suspected chromosome abnormality in first degree relative (parent or sibling)
Maternal age greater than 35
Cancer

3. Common type of dividing nucleated cells or tissues used for cytogenetic analysis
White blood cells (T lymphocytes) from peripheral blood
Amniotic cells (amniocentesis)
Cell of the chorionic villi -- extra-embryonic fetal tissue that form the surface of the chorionic sac (Chorionic Villus Sampling, CVS)
Fibroblasts (skin biopsy)
Cancer cells (tumor biopsy)
Bone marrow cells (bone marrow biopsy for hematological malignancies)

4. Frequencies of Cytogenetic abnormalities and outcomes in human pregnancies
(% of total)
Live Births
(% column 2)
Spontaneous
Abortions
Total
10,000 (100)
8,500 (85)
1,500 (15)
Normal chromosomes
9,200 (92)
8,450 (91.5)
750 (8.5)
Chromosomal abnormalities
800 (8)
50 (6.3)
750 (93.7)
Aneuploidy, unbalanced
rearrangements, others
781 (7.8)
34 (4.3)
747 (95.7)
Balanced rearrangements
19 (0.19)
16 (84)
3 (16)

5. Classification of Human Chromosomes
Satellite:
rRNA coding genes-hundreds of copies
Image from the website of Carol Guze ( - Biology 442, California State Univ., Dominguez Hills

6. Ideogram showing G-banding patterns for human chromosomes at metaphase
The numbering system shown provides a precise description of the location of each band

7. Explanation chromosome of the band numbering system

8. Explanation chromosome of the band numbering system
Giemsa stained chromosome 12 at the metaphase (left) and prometaphase (right) level of condensation from Nature (

9. Properties of chromosome bands seen with Giemsa staining
Dark Bands (G Bands)
Pale Bands (correspond to R bands)
Stain strongly with dyes that bind AT-rich regions such as Giemsa and Quinicrine (Q Banding)
Stains weakly with Giemsa and Quinicrine
Maybe AT-rich
Maybe GC-rich
Condenses early during the cell cycle, but replicates late
Condenses late during the cell cycle, but replicates early
Gene poor
Gene rich
Alu poor but LINE rich
LINE poor, but Alu rich

10. Alternative chromosome staining methods
Q-banding - chromosomes are stained with quinacrine and examined by fluorescence microscopy. The fluorescent Q bands corresponds almost exactly to the G bands.
R-banding – produced by Giemsa staining of chromosomes heated in phosphate buffer. Results in a dark and light banding pattern that is the reverse of that produced by Giemsa. R banding is the standard method for cytogenetic analyses in Europe and in some US labs.
Fluorescence R-banding – untreated chromosomes are stained with acridine orange and examined by fluorescence microscopy. Fluorescent bands correspond to light bands obtained with Giemsa staining.
High resolution banding (prometaphase banding) – G- or R- banding of chromosomes obtained at an early stage of mitosis (prophase or prometaphase) when they are less condensed.

11. High resolution G-banding of the X-chromosome
Ideograms and photo-micrographs of X-chromosomes at various levels of condensation (from left to right, X-chromosomes at metaphase, prometaphase and prophase).

12. High resolution G-banding of the X-chromosome
22.33
22.3
22.32
22.2
22.31
22.2
22.1
22.13
22.11
22.12 21
21.3
21.2
21.1 12 13
13.1
13.2
13.3
21.1 21
21.2
21.31
21.3
21.32
21.33 22
22.1
22.2
22.3 23 24 25
26.1 26
26.2
26.3 27
27.2
27.1
27.3
Ideograms and photo-micrographs of X-chromosomes at various levels of condensation (from left to right, X-chromosomes at metaphase, prometaphase and prophase).

13. Aneuploidies, abnormalities of chromosome number, are the most common type of human chromosomal disorder
1. Autosomal monosomies are incompatible with live births.
2. Only three autosomal trisomies are capable of producing a live birth.
Trisomy 13 (Patau syndrome) – frequency 1/22,700 live births
Trisomy 18 (Edward syndrome) – frequency 1/7,500 live births
Trisomy 21 (Down syndrome) – frequency 1/580 live births
3. Most common sex chromosomal aneuploidies are compatible with life.
a. Females
Turner syndrome – monosomy X (45,X), 1/4000 female births
Trisomy X – (47,XXX), 1/900 female births
Total of all X chromosomal aneuploidies – 1/580 female births
b. Males
Klinefelter syndrome – (47,XXY and 48,XXXY), 1/1000 male births
47, XYY syndrome – 1/1000 male births
Total of all X or Y aneuploidies – 1/360 male births

14. Karyotype of a male patient with Down Syndrome (47,XY + 21)

15. Karyotype of a male patient with Down Syndrome (47,XY + 21)

16. A frequent cause of Aneuploidy is meiotic nondisjunction (failure of chromosomes to detach from each other during one of the two meiotic divisions)
Nondisjunction in meiosis I produces gametes with one maternal copy and one paternal copy of the extra chromosome
Nondisjunction in meiosis II produces a gamete with either two maternal or two paternal copies of the extra chromosome

17. Abnormalities of chromosome structure – unbalanced rearrangements
The presence of an extra copy of a portion of one chromosome results in a partial trisomy, the presence of a missing a portion of one chromosome results in a partial monosomy
Shown: crossover between misaligned homologous sister chromatids during meiosis. 4 gametes possible: 2 normal, one del b, one dup b.
Can also occur between homologous chromosomes that contain long repeated stretches of highly similar DNA sequences

18. Abnormalities of chromosome structure – unbalanced rearrangements
Most common: 46X,iXq
(15% of women with Turner syndrome)
Unstable through mitosis

19. Abnormalities of chromosome structure – balanced rearrangements
Carriers of rearranged chromosomes in which there is no gain or loss of genetic information usually have a normal phenotype unless disrupt essential gene
Carriers of balanced inversions can produce unbalanced offspring
Crossover during meoisis
Possible results of chromosomal segregation
Resulting Gametes:
Normal or balanced
(others not viable)
Normal, balanced or unbalanced

20. Abnormalities of chromosome structure – balanced rearrangements
Carriers of balanced translocations can produce unbalanced offspring
Balanced carrier
Gametes
Gametes
Is usual
seen in 5 to 20% of sperm from balanced translocation carriers

21. Acrocentric chromosomes:
Robertsonian translocations and their possible consequences
Acrocentric chromosomes:
13, 14, 15, 21, 22
Carriers of a Robertsonian translocation are asymptomatic, but can produce unbalanced gametes that result in monosomic and trisomic zygotes. Of the four possible unbalanced zygotes shown here, only trisomy 21 is viable, producing an infant with Down syndrome. A B
45,XY,rob(14;21)
Short arms of acrocentric chromosomes contain long stretches of repeated DNA that encode ribosomal RNA

22. Microdeletion and duplication disorders
(sometimes called Contiguous Gene Syndromes—group of neighboring genes involved)
Disorder
Site
Rearrangement
Repeat
Length (kb)
Type
Size (kb)
Prader-Willi and Angelman syndromes
15q11-q13
Deletion
5000
200
Neurofibromatosis type I
17q11.2
1500 ~
Charcot-Marie-Tooth disease
HNLPP
17p12
Duplication 24
DiGeorge or velocardio-facial syndrome
Cat-eye syndrome
22q11
3000
Williams syndrome
7q11.23
1600
Can result from unequal crossing over between misaligned sister chromatids or homologous chromosomes containing highly similar DNA sequence repeats. The severity of the resulting syndrome often depends on the number of genes affected.
Prader-Willi/Angelman syndromes illustrate the concept of genomic inprinting.
The other disorders in this table are discussed in the textbook. For example, DiGeorge or velocardiofacial syndrome is one of the most common cytogenetic deletions, with a frequency of 1/2000 to 1/4000 live births, and accounts for as many as 5% of all congenital heart defects in the newborn.

23. Spectrum of resolution in chromosome and genome analysis.
The typical resolution and range of effectiveness are given for various diagnostic approaches used routinely in chromosome and genome analysis.

24. Fluorescence In Situ hybridization – one type: Chromosome Painting

25. Detection of trisomies by FISH analysis of interphase chromosomes
Left Panel – 46, XY (X-chromosome green; Y-chromosome red; chromosome 18 aqua).
Center Panel – 47, XX + 18 (X-chromosome green; chromosome 18 aqua).
Right Panel – 47,XX + 21 (X-chromosome green; chromosome 21 red).

26. Detection of deletion of 22q11.2 in DiGeorge syndrome
Green signal, probe to distal segment of the q arm of chromosome 22
Red signal, probe to the segment of the q arm of chromosome 22 adjacent to the centromere
Inset is an interphase nucleus.

27. Chromosomal microarray to detect chromosome and genomic dosage.
Use of assays based on Comparative Genome Hybridization (CGH)

28. Results from a patient with Rett Syndrome
Array CGH analysis:
Results from a patient with Rett Syndrome
Duplication of about 800 kb in band Xq28 containing the MECP2 gene

29. Genome Analysis by Whole Genome Sequencing
Increasingly practical: has become more efficient and less costly

30. Chromosomal and genomic approaches to the diagnosis of trisomy 21
FISH:
Green: autosome
Red: Xsome 21
Whole genome chromosomal microarray
karyotype
Whole genome sequencing: overrepresentation of sequences from chromosome 21
3 copies instead of 2

31. Parent-of-origin effects---Genomic imprinting
Some gene products are harmful at a high dose
One of the two alleles needs to be turned off during development
Depending on the gene, either mom or dad inactivates the gene during gametogenesis
Genomic Imprinting:
One parent passes on an active version of the gene and the other parent passes on an inactivated version
Imprinted state persists in most tissues after fertilization and during postnatal development
Inactivation of imprinted genes: methylation of CG dinucleotides (CpG methylation) in promoter region of the gene
Germ cells:
when female child inherits a paternally imprinted allele from her dad (imprinted to be on or imprinted to be off, depending on the gene), she must reverse the imprint in her germ cells so that she can pass on the maternal imprint to her offspring.
The same is true for males that inherit a maternally imprinted gene from their mom.

32. Fate of the maternal and paternal imprint during passage through the germline in males and females

33. Prader-Willi and Angelman region
Map of imprinted regions in the human genome
Prader-Willi and Angelman region
Prader-Willi and Angelman Syndromes result from multiple causes affecting 15q11-q13.

34. Prader-Willi and Angelman phenotypes
Obesity, excessive and indiscriminate eating habits,
Small hands and feet, short stature, hypogonadism,
Mental retardation
Unusual facial appearance, short stature, severe mental retardation, spasticity, seizures

35. Genes only expressed from maternal copy
Genes only expressed from paternal copy
Chromosomal microarray
(array CGH):
Deletion of region