1

Chromosomal basis of heredity

Lecture one 6th February 2012

2

Why an interest in Clinical Genetics?

• An understanding of genetics is becoming increasingly important for health care practitioners • Rare familiar traits (e.g. cystic fibrosis, haemophilia, phenylketonuria, etc.) have been recognised for some time• Now thought that most major diseases (e.g. cancer, heart disease, diabetes, psychiatric disorders, etc.) have a genetic component

2

3

Introduction to

• We are going to explore various aspects of clinical genetics, including:

chromosomal disorders single gene disorders multifactorial disorders mitochondrial disorders

3

4

Introduction to

• We are going to examine genetic variation at: an individual level, and a population level

• We are also going to study a range of more specialist topics including:

developmental genetics immunogenetics the genetics of cancer

4

5

Review of chromosomal basis of heredity

• Gregor Mendel: is universally acknowledged as the founder of genetics formulated the laws of inheritance in the 1860s but knew nothing about meiosis

• Link between Mendel’s theory and the observed behaviour of chromosomes at meiosis was not made until the beginning of the 20th century• It then became clear that Mendel’s “determinants” (now known as genes) were physical units on chromosomes

5

6

Structure of eukaryotic chromosomes• The nucleus contains linear chromosomes composed of double stranded DNA coiled around histone proteins• The packaging of this complex, called chromatin, varies:

During interphase (G1, S & G2), DNA is relatively loosely associated with the histones, the chromosomes are very long and thin, and hence can only be observed under an electron microscope At the start of nuclear division (mitosis or meiosis), DNA becomes very tightly coiled around the histones (a process known as condensation), hence the chromosomes become much shorter and thicker, and can now be seen under a light microscope

6

7

Cell CycleCell Cycle

copyright cmassengale

8

Karyotype• A karyotype is defined as either the chromosomal constitution of an individual or the representation (as a photograph or diagram) of that chromosomal constitution• The term ideogram is also used for diagrams

8

9

Human karyotype analysis • Conventional karyotyping utilises mitotic metaphase chromosomes• Samples of lymphocyte precursor cells (from bone marrow) or chorionic villi cells (obtained via chorion biopsy at 9-14 weeks gestation) usually contain sufficient dividing cells for direct examination• Most other tissue samples (e.g. T lymphocytes from venous blood, skin fibroblasts, foetal cells shed into the amniotic fluid and obtained via amniocentesis at 16-18 weeks gestation, etc.) require culturing in vitro, a process that can take up to three weeks

9

10

Human karyotype analysis • Dividing cells are arrested at metaphase using a spindle inhibitor such as colchicine • Cells are then spread on a glass slide in the presence of a hypotonic saline solution that causes the nuclei to rupture and the chromosomes to separate • Chromosomes were observed under the light microscope as early as the mid 19th century• Human cytogenetics dates from 1956 when Tjio and Levan established that the normal human chromosome number in somatic cells was 46 with:

22 pairs of autosomes, and 1 pair of sex chromosomes (XY in males and XX in females)

10

11

Human karyotype analysis • Traditionally, chromosomes were cut out from a photograph and glued on to card• This stage is now largely undertaken electronically utilising digital images• Whichever method is used, the 22 pairs of autosomes are arranged according to:

length, from longest (chromosome 1) to shortest (chromosome 22), and position of centromere

11

12

Human karyotype analysis • Chromosome arms are respectively designated p (for short) and q (for long):

• By convention, p arms are shown uppermost in karyotypes• The p and q arms of metacentric chromosomes have been designated by international agreement

12

13

Human karyotype analysis Human chromosomes were originally divided into seven groups: A (1-3), B (4-5), C (6-12 plus X), D (13-15), E (16-18), F (19-20) and G (21-22 plus Y):

13

14

Human karyotype analysis • Staining procedures are normally used to visualise chromosomes • As these techniques improved, initially individual chromosomes could be identified, and then smaller and smaller chromosomal sections could be recognised• The most common procedure, G-banding, uses the protease trypsin to denature chromosomal proteins followed by treatment with Giemsa• As Geimsa binds differentially, characteristic patterns of dark and light bands result• The darker stained G-bands are rich in adenine and thymine while the lighter stained G-bands are rich in guanine and cytosine• The latter have been found to contain more genes 14

15

Ideogram of human G-banded chromosomes

15

16

G-banded human chromosomes

• Such banding has greatly facilitated the identification of individual chromosomes and their subdivision into a series of regions• In current nomenclature:

each chromosome arm is divided into regions numbered outwards from the centromere (p1, p2, etc.) then each region is subdivided into bands also numbered outwards from the centromere (p11, p12, etc.)

• Thus on the short arm of chromosome 2, 2p12 lies centromeric to 2p13 but telomeric to 2p11

16

17

Regions and bands on chromosome 2(at low resolution)

17

18

Regions and bands on chromosome 7(at a range of resolutions)

At higher resolutions,further subdivisionsare sometimes used(e.g. 7q31.32 refersto chromosome 7,long arm, region 3,band 1, sub-band 3,sub-sub-band 2, etc.)

18

19

Other banding techniques

Other less commonly used techniques include:• Q (quinacrine) banding• C (centromeric) banding• NOR (nucleolar organiser region) staining which visualises the satellite stalks present on the p arms of all acrocentric chromosomes except Y

19

20

Fluorescence in situ hybridisation (FISH) • Is a technique where one or more fluorescently labelled DNA probes are applied directly to the chromosomes• Unlike conventional karyotyping, interphase chromosomes can be analysed and hence a rapid diagnosis of a suspected genetic abnormality can be obtained• Individual genes can be localised and abnormalities beyond the resolution of the light microscope can be seen• Applications of FISH include the detection of:

submicroscopic deletions translocations (such as the Philadelphia chromosome and translocation of the SRY locus from Yp11 often to X) trisomy (using centromeric probes)

20

21

Multicolour (M) - FISH (also known as spectral karyotyping)

• The so-called chromosome painting technique uses the same fluorochrome to label a range of sequence probes for an individual chromosome• Then, if each of the 24 different chromosomes (1-22, X and Y) are labelled with a different fluorochrome, the whole karyotype can be observed:

21

22

Multicolour (M) - FISH (also known as spectral karyotyping)

• The main application is the visualisation of chromosomal rearrangements that occur during the development of some types of cancer

• Note that M-FISH does not as yet offer the degree of resolution provided by conventional chromosome analysis• Hence FISH and M-FISH are currently used to supplement conventional analysis rather than to replace it

22

23

Chromosomal mutation • Two broad categories of mutation occur:

point mutation where only a single base or a few bases are altered chromosomal mutation where an entire chromosome or a large region of a chromosome is altered

• Chromosomal mutations can be detected by karyotype analysis • Chromosomal mutations are of two main types:

changes in chromosome number chromosome rearrangements

23

24

Changes in chromosome number• May arise from:

euploidy, the loss or gain of whole sets of chromosomes – so euploid cells contain exact multiples of the haploid number (n) aneuploidy, the loss or gain of individual chromosomes – so aneuploid cells have incomplete sets of chromosomes

Reminder: n = 23 in humans

24

25

Euploidy: monoploids (n)

• The term monoploid is used for euploid mutants with only one set of chromosomes

• Monoploids arise in diploid species via parthenogenesis, viz. the development of an egg without fertilisation• Monoploid animal embryos (including humans) do not develop far

25

26

Euploidy: polyploids (>2n)

• Polyploids have more than two sets of chromosomes • For example:

triploids (3n) have three sets of chromosomes tetraploids (4n) have four sets of chromosomes

26

27

Euploidy: polyploids (>2n) • Mature polyploid animals are rare• Approximately1% of human conceptions are triploid but most die before birth• Those that are born alive (1 in 10,000 live births) do not survive long

27

28

Euploidy: polyploids (>2n)

Polyploidy can occur spontaneously in a number of ways:• An egg may be fertilised by more than one sperm, a condition known as dispermy. This is the most common cause of triploidy • Failure of reduction division at meiosis will led to diploid gametes. Fertilisation of such a diploid gamete with a normal haploid gamete will then result in triploidy • In females, one of the polar bodies (usually non viable products of meiosis) may fuse with the ovum to produce a diploid gamete. Fertilisation with a normal haploid sperm will then result in triploidy

28

29

Aneuploidy• Aneuploidy arises as a consequence of non-disjunction of chromosomes (i.e. failure to separate properly) at mitosis or meiosis, respectively resulting in mosaic formation or abnormal gametes• Aneuploids fall into a number of groups, for example:

29

30

Human aneuploidy• All types of autosomal monosomy are lethal• Approximately 4% of human conceptions are trisomic• While the gain of an autosome is not usually compatible with survival beyond early pregnancy, the following are observed in live births:

trisomy 13 (Patau syndrome) trisomy 18 (Edwards syndrome) trisomy 21 (Down syndrome)

30

31

Down syndrome (trisomy 21)

31

32

Down syndrome (trisomy 21)

32

33

Down syndrome (trisomy 21)

33

34

Human aneuploidy• The presence of an extra sex chromosome does not apparently compromised embryonic survival and the following sex chromosome trisomics are observed in live births:

47,XXY (Klinefelter syndrome) 47,XYY (XYY syndrome or double Y syndrome) 47,XXX (triple X syndrome or trisomy X)

• Loss of an X chromosome generating 45,Y is lethal• Loss of either an X or a Y chromosome generating 45,X (Turner syndrome) is observed in live births but most such embryos are lost during pregnancy

34

35

Klinefelter syndrome (trisomy XXY)

35

36

Klinefelter syndrome (trisomy XXY)

36

37

Turner syndrome (monosomy X0)

37

38

Turner syndrome (monosomy X0)

38

39

Summary of human aneuploidy

39

40

Chromosome rearrangementsSeveral different classes of structural rearrangement are recognised: • Deletion

• Duplication

• Inversion

40

41

Examples of human chromosome rearrangements

• Deletion: Cri du chat syndrome results from a deletion involving the terminal region of chromosome 5p Both Angelman and Prader-Willi syndromes result from a microdeletion involving chromosome 15q

• Duplication A small proportion of Down syndrome cases result from the duplication of the q arm of chromosome 21. Such patients have 46 chromosomes, including one normal 21 and one "isochromosome" (i.e. a chromosome with the extra copy of the q arm replacing the p arm)

41

42

Chromosome rearrangementsStructural rearrangement also include several types of translocation: • Reciprocal translocation

• Non reciprocal translocation

• Robertsonian translocation

42

43

Human chromosome translocations

The carriers of either reciprocal translocations or Robertsonian translocations are at risk of having children with unbalanced chromosome complements:• Familiar Down syndrome can be caused by a Robertsonian translocation between chromosomes 14 and 21•The risk of a carrier producing a child with Down syndrome varies with:

the chromosome pairing arrangements during meiosis which parent is the carrier - 10-15% risk for female carriers and 1-2% risk for male carriers

43

4444

4545

46

Other outcomes of human chromosome rearrangements

• Some 20% of cancers are thought to result from inversions, translocations and transpositions as such chromosomal rearrangements may change changing the location of growth regulating genes• For example, the so-called Philadelphia translocation between chromosomes 9 and 22 is characteristic of chronic myeloid leukaemia

46

47

Clinical cytogenetics Cytogenetic investigations are undertaken in the following circumstances: • suspected chromosomal abnormality• infertility or multiple miscarriages• stillbirth or neonatal death• multiple congenital anomalies and/or developmental retardation• undiagnosed mental retardation• disorders of sexual function• certain malignancies

47

48

Clinical cytogenetics

• Total chromosome count is normally determined in 10-15 cells (increased to a minimum of 30 cells if mosaicism is suspected) • A detailed G-banding pattern analysis is undertaken in 3-5 cells• The formal report utilises standard abbreviations that can be interpreted by health care practitioners • Most cytogenetic laboratories also provide written guidance where findings are complex

48

49

Abbreviations used in cytogenetic reports p short armq long armcen centromeretel telomeredel deletiondup duplication + gain of

chromosome- loss of

chromosome

i isochromosomeins insertion inv inversionr ringt translocationrcp reciprocal

translocationrob Robertsonian

translocationmos mosaicism

50

Examples of karyotype reports

46,XX Normal female46,XY Normal male45,X Female with Turner syndrome47,XY,+21 Male with Down syndrome47,XXY Male with Klinefelter syndrome69,XXX Triploid female46,XX,del(5p) Female with cri-du-chat

syndrome

51

Examples of karyotype reports (cont)

45,XX,rob(14;21)(q10;q10)Female with a balanced 14;21 Robertsonian translocation (by convention the positions of the centromeres are designated as q10;q10)

46,XX,rob(14;21)(q10;q10),+21Female with an unbalanced 14;21 Robertsonian translocation resulting in Down syndrome

52

Karyotypes observed with Down syndrome

Chromosome abnormality Frequency

Trisomy 21

Maternal origin Paternal origin

95%

90%5%

14/21 translocation up to 4%

Isochromosome 21 up to 1%

Mosaic trisomy 21 < 1%

53

Fate of onemillion implantedhuman zygotes