The Patient: Cytogenetics

What is cytology? What is cytogenetics? the biology of the formation, structure, and function of cells. Cytogenetics deals with the constituents of the cell involved in heredity, such as chromosomes (chromosome theory of inheritance). It offers another level of genetic analysis. It was created in 1903, combining cytology and genetics. What are chromosomes made up of? How does chromosome appearance change during mitosis? Double helix DNA is wound around histones to form a nucleosome. The nucleosomes are compacted and supercoiled to form chromatin, and then a chromosome. The chromosomes are less condensed during interphase, when most genes are transcriptionally active. The spatial organisation of the chromosomes is important. During mitosis, the chromosomes become condensed. What is karyotyping? A test used to examine chromosomes in a sample of cells. What are metaphase chromosomes classified into in karyotyping? Metaphase chromosomes are classified in terms of number and size of chromosomes, morphology of chromosomes, banding pattern, regions of heterochromatin and euchromatin. What stains are used in karyotyping? Why are stains used? To aid karyotyping, chemical stains such as quinacrine (Q banding) and Giemsa (G banding) can be used to differentially stain chromosomes to reveal species' specific banding patterns. 1 / 12 Why are heterochromatin and euchromatin important? How can you tell the difference between regions of heterochromatin and euchromatin? Heterochromatin and euchromatin are important in controlling gene expression in different cells. They show differential staining. What are the different types of heterochromatin? - Constitutive heterochromatin is highly condensed, mostly highly repetitive DNA, arranged in tandem arrays, very few genes. - Facultative heterochromatin is also highly condensed but can be converted to euchromatin. What is euchromatin? Euchromatin is not highly condensed, it is where most genes are located. Two copies of an X chromosome in females and one in males presents a dosage problem. What is the heterochromatin inactivation To compensate, an X chromosome is of the X chromosome? inactivated (in every female cell) by become facultative heterochromatin. The inactivated X is referred to as a Barr body. - Used distilled water not saline solution How was human karyotyping previously - Dropping the cells to burst nuclei done? - Spindle-poison colchicine used to arrest at mitotic metaphase. How is karyotyping done nowadays? - Culture blood cells and add colchicine to stop mitosis at metaphase. - Spin down cells in a centrifuge and discard supernatant. - Add hypotonic salt solution (low salt) and resuspend the cells. - Add fixative (preservative) to cell suspension, add one drop to the slide and then dry and stain. - Giemsa staining reveals distinct banding patterns unique to each chromosome and species. Each band has a unique 2 / 12 identifier number. The different ends of the centromere are called arms, and the end of the chromosome is called the telomere. Categorised according to the length of the arms and centromere location. - Metacentric chromosomes - Submetacentric chromosomes - Acrocentric chromosomes What are the different human chromosome morphologies and how are they categorised? What is the long arm and short arm abbreviated as? long arm - q short arm - p Where is the region 15q7? Chromosome 15, long arm, band 7 What are metacentric chromosomes? Metacentric chromosomes have short and long arms of roughly equal length with the centromere in the middle. What are submetacentric chromosomes? Submetacentric chromosomes have short and long arms of unequal length with the centromere more towards one end. What are acrocentric chromosomes? Acrocentric chromosomes have a centromere very near to one end and have very small short arms. They frequently have secondary constrictions on the short arms that connect very small pieces of DNA (stalks and satellites) to the centromere. The stalks/satellites contain genes that code for ribosomal RNA. - x is the basic number of different chromosomes in a single haploid set (this is What is x, n and 2n when talking about 23 in humans). chromosome numbers? - n is the gametic number of chromosomes (this is 23 in humans). - 2n is the number of chromosomes 3 / 12 found in the zygote and the soma (this is 46 in humans). Ploidy is the number of sets of chromosomes in a cell, or in the cells of an organism. What is ploidy? What are polyploid organisms? Polyploid organisms have more than 2 sets of chromosomes. It is common in plants, but rare in animals (limited to certain beetles, earthworms, salamanders, fish). In humans, it is pathological and leads to early death. What are a high proportion of neonatal abnormalities due to? (clinical cytogenetics) congenital chromosome anomalies, which can lead to birth defects. What are congenital anomalies? Congenital anomalies (birth defects, congenital disorders or congenital malformations) are structural or functional anomalies present at or before birth but occurred during intrauterine life. When are congenital anomalies identified? The condition may be identified prenatally, at birth, or later in infancy if less apparent such as hearing loss. What plays an important role in many congenital anomalies? What are some examples? Gene mutations - Trisomy 13 (Patau syndrome) - Trisomy 18 (Edwards syndrome) - Trisomy 21 (Down syndrome) FISH (Fluorescent in situ Hybridisation) What are the clinical indicators for chro- is a cytogenic technique used to detect mosomal analysis? and locate a specific DNA sequence on a chromosome. 4 / 12 How does FISH work? A DNA probe (specific DNA sequence) is tagged to a fluorescent dye (such as fluorescein), and it will bind to the matching sequence within the set of chromosomes. What can be investigated by using the FISH technique to observe chromosomes? This technique can be used to highlight specific regions of chromosomes and to look at multiple congenital abnormalities, abnormality in sexual development, infertility, recurrent miscarriage, unexplained stillbirth, learning difficulties, and malignancy (such as inherited retinoblastoma caused by deletions in the region of a chromosome or Burkitt lymphoma caused by a chromosome translocation). What do chromosome disorders have a major contribution to? - Morbidity - Pregnancy loss - Childhood disability - Malignancy - Mortality What are some examples of changes in chromosomes (general abnormalities)? - Constitutional changes (germ line in origin) - Somatic changes (certain cancers) - Numerical changes - Structural changes Can be balanced where there is no loss or gain of genetic material, or it can What does it mean if a chromosome abbe unbalanced where genetic material normality is balanced or unbalanced? is lost or gained with serious clinical effects. - Polyploidy (Triploidy and Tetraploidy) What are some examples of numerical - Aneuploidy (having an abnormal number of chromosomes in a haploid set abnormalities in chromosomes? Trisomy, Monosomy and Tetrasomy - four copies of a particular 5 / 12 chromosome instead of 2. - Mixoploidy (two cell lines in one organism, cells with different numbers of chromosomes) What is triploidy? - Presence of an additional set of chromosomes in the cell for a total of 69 chromosomes rather than the normal 46 chromosomes per cell. - Examples include 69,XXX, 69,XXY, and 69,XYY. This makes up 2-3% of all human pregnancy, most will not go to term and those that do die shortly after birth. What is tetraploidy? Four copies of each chromosome in the cell instead of two, resulting in a total of 92 chromosomes in each cell. An example is 92,XXXX, and this makes up 1-2% of miscarriages. How does triploidy (tripronuclear zygotes) originate? How do polar bodies normally form in humans? - Digyny - extra chromosome set from the mother (diploid oocyte) and normal (haploid) spermatozoon - Diandry - normal (haploid) oocyte, and a diploid spermatozoon, or dispermic fertilisation (polyspermy) A polar body is the byproduct of an oocyte meiotic division. The first polar body (with almost no cytoplasm) degenerates. The second division produces a second polar body, which splits off and remains adjacent to the oocyte, until it also degenerates. Polar bodies remove a set of chromosomes, to make the oocyte haploid. 6 / 12 Polar body is retained in the egg so that How do polar bodies abnormally form in the gamete has the full diploid genome. humans leading to digyny? When it is fertilised by the haploid sperm, it generates the triploid zygote. What is trisomy? The presence of an extra chromosome (47 chromosomes) and is usually embryonic or foetal lethal. What are some examples of trisomy? - Patau syndrome (trisomy 13) - Edwards syndrome (trisomy 18) - Down syndrome (trisomy 21) - Trisomy 16 (common in spontaneous miscarriage) - Klinefelter syndrome, 46 XXY (presence of additional sex chromosome has relatively mild phenotypic effects) - Extra digit on hands or feet (polydactyly) - Can also be associated with neurological problems, mental and motor deficienWhat is Patau syndrome (trisomy 13)? cies, eye, heart, and spine defects. - 80% die within the first year, affects 1 out of every 10,000 new borns. Most cases result from the events in the sperm or egg (not inherited). What is Edward's syndrome (trisomy 18)? - Mental retardation, breathing and feeding difficulties, delayed growth, malformations of the kidneys, intestines, and heart. - 95% miscarry, 1 out of every 3,000 newborns, 90% die within the first year. Most cases are not inherited. - Decreased muscle tone, a flat face, eyes slanting up, irregular shaped ears, ability to extend joints beyond the usuWhat is Down syndrome (trisomy 21)? al, large space between the big toe and neighbouring toe, large tongue relative to mouth, intellectual disabilities. 7 / 12 They can also suffer from heart disease, Alzheimer's disease, and leukaemia. - 1 out of every 1,000 newborns. How does trisomy originate? What is monosomy? Non-disjunction can occur at first or second division, two chromosomes end up in the same gamete. It is not usually inherited, it occurs in the formation of the gamete. The absence of one chromosome (45 chromosomes). It is almost always incompatible with survival to term, except for Turner syndrome (45, X) where there is a missing X chromosome. People with Turner syndrome typically experience gonadal dysfunction which results in absence of menstrual cycle and sterility. Deletion, inversion, duplication, insertion, ring, reciprocal translocation, and Robertsonian translocation. What are some examples of structural abnormalities of chromosomes? How can you identify translocations in chromosomes? - Detailed chromosome banding - FISH In a reciprocal translocation, there is an exchange of two regions (common in What occurs in a reciprocal transloca- chromosome 11 and 12). There is no tion? When can there be consequences? gain or loss of genetic material but can have consequences if the breakage is at a coding region of DNA/within a gene 8 / 12 but risks of an unbalanced chromosome complement. This occurs in 1 of 500 in the general population. This can affect meiosis, as there can be different types of gametes (3 outcomes). - Alternative segregation can occur which yields normal or balanced haploid complement. The two chromosomes segregate normally, leading to the same genetic material in both new cells. - Adjacent-1 segregation yields unbalWhat can a reciprocal translocation af- anced haploid component. One normal chromosome and one translocated chrofect? mosome both move to the same pole and not opposite sides. - Adjacent-2 segregation yields unbalanced haploid component. Two translocated chromosomes move to the same pole and not opposite sides. - Segregation in meiocytes with unbalanced reciprocal translocations can yield gametes with significant chromosome What are the consequences of reciproimbalance. cal translocations in meiosis? - On fertilisation, this leads to early pregnancy loss or the birth of an infant with multiple abnormalities. A Robertsonian translocation results from the breakage of two acrocentric chromosomes (13, 14, 15, 21, and 22). What is a Robertsonian translocation? This occurs in 1 of 1000 people and most commonly involves chromosome 13 and 14. The short arms are lost but they do not usually contain any genetic material. 9 / 12 During meiosis in individuals with a translocation, the translocated chromosome pairs with another chromosome 14 or 21 during meiosis I. This can lead to the formation of two types of gametes: Gametes with the translocated chromosome and a normal chromosome 14 or 21 (which can segregate to form What are the consequences of Robert- lethal gametes, gametes that can develop Down's and carriers). sonian translocations in meiosis? Gametes with a normal chromosome 14 or 21, but lacking the translocated chromosome (normal). Down syndrome can result due to Robertsonian translocations. It is imporIf a child has Down syndrome, what can tant to screen parents of a child with you do if parents want to have further Down syndrome since if one of the parchildren? ents has a Robertsonian translocation of this type, there is a risk of having further affected children. Monosomy for a fragment of chromoWhat are chromosome deletions? When some. If more than 2% of a haploid genome is lost, this can have a lethal does it become lethal? outcome. What are the two types of location of chromosome deletions? Terminal (at the end) or interstitial (the middle of the chromosome). Genetic material lost can be lost leading to a loss of function, but this depends What does loss of genetic material deon amount of fragment lost, number of pend on in a chromosome deletion? genes in fragment, and how important the genes were. 10 / 12 - Large deletions that can be observed under the microscope after conventional chromosome staining (Wolf-Hirschhorn, Cri du chat syndromes). What are the two levels of chromosome - Sub-microscopic deletions only detectdeletions and how can they be identied by high resolution prometaphase cyfied? togenetics augmented by FISH analysis or array CGH (Microarray Comparative Genomic Hybridisation), such as Prader-Willi and Angleman syndrome) What is Wolf-Hirschhorn syndrome caused by? a deletion of genetic material near the end of the short (p) arm of chromosome 4. What is Prader-Willi syndrome caused some missing genetic material in a group by? of genes on chromosome number 15. - Pericentric inversion (involving the cenWhat are the two types of chromosome tromere of a chromosome) inversion? - Paracentric inversion (involving just the arms of a chromosome) This is balanced and rarely causes problems in an individual unless a Why are chromosome inversions usually break point has disrupted an important balanced? gene. The problems arise when unequal crossovers occur in heterozygotes. When two chromosomes (one normal and one pericentric inverted) try to pair up correctly during fertilisation, this leads to contorted conformations and strucWhat happens in crossovers with a peritures which introduce a lot of tension. centric inverted chromosome? This can lead to breakage. Fertilisation of abnormal gametes with these inversions leads to early miscarriage or a baby with multiple abnormalities. 11 / 12 What happens with crossovers with a paracentric inverted chromosome? When an abnormal chromosome with a paracentric inversion is crossed over with another chromosome, segments of genetic material can be missing or duplicated when chromosomes try to pair up. This can result in a loss of genetic material, due to the absence of the centromere, affecting the proper alignment of chromosomes during cell division. Centromeres are what connect to the tubule system. 12 / 12