Joseph Schlammadinger retired associate professor

1. Joseph Schlammadinger retired associate professor Lectures in March, 2013 (revised versions)

2. Note and don’t forget, please • Facts, their description, organization and interpretation within one chapter of a given science–in our case: Genetics–may be different as taught by different lecturers, or seen in different sources (e.g. textbook vs. lecture, and so on). The essence, however, is the same even if different terms, words, expressions are actually used, provided the students understand and use them correctly. • This formula is true to exam questions, too.

3. CYTO- GENETICS 2013

4. Morphologically (microscopically) identifiable chromosomes are seen only in eukaryotic organisms Cytogenetics is the science of chromosomes, elements of genetics which can be analyzed cytologically. (Better in LM than in EM.) ONE CHROMOSOME = ONE DNA MOLECULE The linear DNA molecule runs form one end of the chromosome (telomere) to the other end. It is organized into chromatin (with histones and other proteins), in a form of multiple coils. The physical length of a metaphase chromosome is approximately 1/10,000 of the total length of the DNA making that very chromosome. The sequence of the genes within one DNA molecule, i.e. within a given chromosome is determined, and is characteristic and specific to species. (See details in genetics: linkage, linkage groups, etc., and the results of the Human Genome Program.)

5. (G0)  G1  MS  G2 Phases of the cell cycle: G1, S and G2 = interphas Chromosomes are not visible individually*. M = mitosis. Individual chromosomes are visible, the best in the metaphase. * Novel methods, however, may show (and even identify) the individual chromosomes or part(s) of them in interphase, too. (See FISH and next slide.)

6. Arrangement of chromosomes in an interphase nucleus. (Computer reconstruction in false colours.)

7. MITOSIS (first half)

8. MetaphaseMITOSIS (second half  cytokinesis)

9. Metaphase chromosome as seen in the electron microscope

10. THE NUMBER*, SHAPE, relative SIZE, BANDING PATTERN and GENE SEQUENCE (= order of loci**) of CHROMOSOMES is a characteristic standard for each species. Morphological (light microscopic) features of chromosomes of each species are summarized in the IDEOGRAM, most conveniently in diagrammatic representation. The chromosomes of a given individual are evaluated by comparing them to the ideogram of the very species concerned. *Higher order eukaryotes are typically diploid organisms. The normal (diploid) human chromosme number is 2n = 46. ** Locus (latin) = place, location (plural: loci). In genetics: the location of a gene in question within a specified chromosome. Variable stained

11. LIGHT MICROSCOPIC INVESTIGATION OF HUMAN METAPAHSE CHROMOSOMESI. TRADITIONAL PROCEDURE 1/ Dividing cells (predominantly: in vitro cell culture, e.g. PHA [phytohemagglutinin] stimulated T lymphocytes). 2/ Accumulation in the metaphase: aided by colchicine treatment. 3/ Swelling in hypotonic solution (0.075 M KCl). 4/ Fixing. 5/ Spreading on specimen slides. 6/ Staining: Giemsa’s solution. 7/ Light microscope: metaphase plate. 8/ Photography. 9/ Print. Cut out images of individual chromosomes and arrange them according to the principles of the ideogram (Denver nomenclature). 10/ The result is an ordered (arranged) karyogram. Evaluate and 11/ establish karyotype.

12. Some comments on terminology English (mainly USA)European (printed photo of a) metaphase spread not ordered karyogram karyotype (in photographic presentation)ordered karyogram karyotype (symbols) karyotype (symbols)

13. SHAPES OF CHROMOSOMES, IA: metacentric, B: submetacentric, C: acrocentric.1: sister chromatids, 2: centromere, 3: short arm, 4: long arm, 5: satellite, 6: secondary constriction.A (not ordered) karyogram

14. Shapes of chromosomes, II (for instance in mouse)

15. PRINCIPLES OF KARYOGRAM ARRANGEMENTS(diploid human cells) The Denver system. Order the chromosome images according size: the biggest is the first. In the case of same size pairs the more metacentric comes first. The homologous pairs, which are identical in both sexes, are numbered from 1 to 22. These are called autosomes. Sex chromosomes are denoted with X and Y. They are not homologs, contain different genes, but in meiosis of the male they behave as homologs in the reduction division. Chromosomes also have been grouped: A (1-3) = big metacentrics, B (4-5) = big submetacentrics, C (6-12) = medium size submetacentrics, D (13-15) = big acro-centrics, E (16-18) = small submetacentrics, F (19-20) = small metacentrics, G (21-22) = small acrocentrics. On that basis X belongs to group C, Y to group G.

16. ARRANGED KARYOGRAMSFEMALE MALE

17. Description of karyotypes with conventional symbols: 46,XX = altogether 46 chromosomes, among them two X, normal female karyotype. 46,XY = altogether 46 chromosomes, among them one X and one Y, normal male karyotype. In English speaking countries (and in general: in English publications) usually the image of a–representative, characteristic–ordered karyogram is given as a karyotype. (The use of the term karyogram is only occasional.) (See further details below.)

18. LIGHT MICROSCOPIC INVESTIGATION OF HUMAN METAPAHSE CHROMOSOMESII. BANDING TECHNIQUES 1/ Dividing cells (predominantly: in vitro cell culture, e.g. T cells). 2/ Accumulation in the metaphase: aided by colchicine treatment. 3/ Swelling in hypotonic solution (0.075 M KCl). 4/ Fixing. 5/ Spreading on specimen slides. (Pretreatment for banding.) 6/ Staining: Giemsa’s solution or quinacrine (a fluorescent dye). These give G bands and Q bands, respectively. (There are also other bands, like C, R and T, developed by special staining procedures.) 7/ Conventional light or fluorescent microscope. 8/ Photography. 9/ Print. Cut out images of individual chromosomes and arrange them according to the principles of the banded ideogram (Paris nomen- clature). 10/ The result is an ordered (arranged) karyogram. Evaluate and 11/ describe karyotype.

19. Q-bands and G bands on chromosomes, and (Paris) ideogram

20. There are other banding procedures, too, resulting in C, R and T bands, respectively. Today the Giemsa (G) banding technique is mostly used (G+ = dark staining, G– = light staining). Chromosome arms are divided in regions and these are numbered 1, 2, 3, and so on in both p and q directions, starting from the centromere. The bands within the regions are numbered according to the same rule. In quinacrine (Q) banding Q+ = intense fluorescence, Q– = faint fluorescence. Q+ = G+, Q– = G–. A number of new high resolution banding techniques have also been developed which allow better longitudinal resolution within a chromosome. Human chromosome #1, standard (left) and results of two high resolution bandings. 

21. NUMERICAL ABERRATIONS OF HUMAN CHROMOSOMES

22. NUMERICAL ABERRATIONS,POLYPLOIDIEStriploidy (3n = 69) tetraploidy (4n = 92)

23. Human numerical chromosome aberrations,POLYPLOIDIES All these are mentioned as euploidy, the actual chromosome numbers are exact multiples of n, i.e. of the haploid set.

24. Please note. Triploidy as well as tetraploidy are lethal conditions in humans, they result in spontaneous abortion rather than in stillbirth. The traditional nomenclature defines these conditions, however, as euploidy: they are exact multiples of n. (The haploid chromosome set is represented by n. In Homo sapiens n = 23. Triploidy = 3n, tetraploidy = 4n.) “Eu” as first syllable in some compound words means “real”, “right”, “good”, “regular”, “self evident”, and so on. In plants polyploidy may contribute to the development of desirable traits in agriculture (higher yield, better resistance, and so on). The majority of wheat (corn) sorts in production are hexaploid (6n = 42). In that case we speak about allopolyploidy, where the three originally diploid chromosome sets come from three different (closely related) ancestors. In the animal world, in vertebrates polyploidy is compatible with life–if at all–in salamanders (amphibian); in higher order animals that is lethal, like in humans.

25. Human numerical autosome aberrations (anomalies),aneuploidies (Euploidy: see earlier. “an-” = privative prefix. Aneuploid = non-euploid.The chromosome number is not exact multiple of n.)

26. CAUSES OF ANEUPLOIDIES • Nondisjunction. Homologous chromosomes or sister-chromatids fail to separate from each other. It results in hyper- orhypohaploid gamete, or in hyper- and/or hypodiploid somatic cells. Occurs either in a/ meiosis, when all the cells of the offspring are aneuploid, or in b/ mitosis, where it creates chromosome mosaicism, that is, in one individual two (or more) cell types (cell lines) can be ascertained with different chromosome numbers. (For instance 46,XX / 47,XX,+21.) The extent and severity of the symptoms depend on the ratio of normal / aneuploid cells, on one hand, on the other, on the tissues (organs) involved. • Delay (late arrival) of a chromosome. In the telophase of the cell division a chromosome cannot reach the newly forming nucleus, remains in the cytoplasm, where it will be destroyed. This phenomenon creates monosomy, i.e. hypodiploidy. May occur in mitosis and in meiosis as well. >2n = hyperdiploid, <2n = hypodiploid. The latter is rare because of the low viability of monosomic cells.

27. NONDISJUNCTION… …of X chromosomes in the first (left) or in the second (right) female meiotic division. Please observe. If the first meiotic division is abnormal, the resulting oocyte (mature egg cell) contains the two X homo-logs. If a nondisjunction occurs in the second meiotic division, sister chromatids remain together, which results in the presence of two identical X chromosomes in the egg cell (apart from the effects of crossing over).

28. MITOTIC NONDISJUNCTION The monosomic cell is non-viable,at least in the majority of the cases.

29. MOSAICISM An individual organism (from one zygote) is composed of two (or more) genetically different types of cells. In this case: chromosome mosaicism, e.g. 46,XY/47,XY,+21. CHIMERISM An individual organism contains genetically different cells which derive from two zygotes. In our example: chromosomal chimerism, e.g. 46,XX/46,XY. Possible sources of chimeras Two egg cells fertilized by two sperm cells, andthe two zygotes form one preembryo. Essentially the same, one cell, however, is a fer-tilized polocyte (fed by the fertilized egg cell). Dizygotic twins may exchange cells (e.g. bonemarrow stem cells) via their common placenta. Green signal = X chromosome. Organ transplantation: 46,XY ► 46,XX. Red signal = Y chromosome. Blue= nucleus

30. DOWN SYNDROME, I

31. What is a syndrome? A group of symptoms and signs, which, when considered together, characterize a disease or lesion. It is not necessary to see all diagnostic signs in one patient, some 3-4 very typical leading symptoms may already define a syndrome. In this cytogenetics lecture you will see: Down syndrome, Pätau syndrome, Edwards syndrome, Turner syndrome, Klinefelter syndrome, and Cri du chatsyndrome (cat cry disease). You need not to learn the symptoms and signs of the syndromes listed above (those are dealt with in detail by Clinical Genetics), but you are supposed to mention the most important characteristic(s), one or two, in order to show that you know what you are speaking/writing about.

32. Down syndrome, II

33. Down syndrome as trisomy 21 Karyotype: 47,XX,+21 or 47,XY,+21, that is trisomy 21. If the supernumerary chromosome #21 is present in all the cells of a Down syndromic individual, we deal with a form of meiotic origin. (See the characteristic symptoms and signs in the next figure.) In the case of mosaicism (which is very rare), the symptoms are strikingly variable and diverse, e.g. no mental retardation in the presence of well visible facial signs (or vice versa), and so on.

34. Symptoms and signs in Down syndrome     Increased liability to leukemiaIncreased liability to infections 

35. Down syndrome and maternal age Incidence of meiotic trisomy 21 in relation to maternal age at birth. Pregnant women above 35 are at increased risk to have a Down syndromic baby, they should be offered a pre-natal diagnosis. (The risk of other aneuploi-dies is also higher.) Don’t forget: the majority (about 80%) of Down syndromic child-ren are born to mothers <35 years.

36. Maternal age and risk of Down syndromic birth(international statistical data) Maternal age Maternal age Maternal age

37. Do we have any means to prevent birth of Down syndromic babies? Yes, termination of pregnancy with a 21 trisomic embryo/fetus. Not legal in all countries. Where permitted on the basis of medical indication, one needs well established diagnosis. How can a reliable diagnosis be made? By karyotyping only. Sampling for that, however, is an invasive method. Based on the statistical data cited above, is the maternal age >35 yr the only indication of a prenatal diagnostics? And why 35? The latter is a compromise. Risk of “spontaneous” abortion after sampling vs. risk of birth of Down syndromic child. Above 35 the latter is higher. Are there some non-invasive methods at hand indicative of Down syndromic pregnancy, if present, in maternal age groups <35 yr? First: in utero ultrasound investigation of the embryo. Second: determination of biochemical markers in maternal blood, which might be different in normal vs. Down syndromic pregnancies. If these investigations disclose some elevated risk of the birth of a Down syn-dromic child, chromosome diagnosis has to be made.

38. Down syndrome diagnosis and screeningAssesment of risk in utero Praenatal chromosome analysis – reliable diagnosis (1)Chorionic villus sampling (CVS) / (2) Amniocentesis Screening in (early) pregnancy Ultrasound. Determination of the size of nuchal translucency (NT). Biochemical marker investigations in maternal blood. They are non-invasive methods (taking venous blood is generally assumed harmless). The evaluation is statistical, if more markers are investigated, they can be indicative of the eventual presence of a Down syndromic embryo/fetus in the maternal womb. A combina-tion and comparison of the first and second trimester findings are evaluated. Main biochemical markers used in screening protocols: Alpha-fetoprotein (AFP)* Non conjugated (unconjugated) oestriol (uE3) Inhibin A (INH-A), a peptide hormone (of two subunits), inhibits FSH** excretion. Pregnancy-associated plasma protein (PAPP-A) Free and/or total human choriogonadotrophin (hCG) * Very important in prenatal diagnostics of neural tube defects (NTD), see there.** Follicle stimulating hormone.

39. ◄ Sketch of amniocentesisFetal cells in the amniotic fluid are obtained. (These cells as well as the amniotic fluid are/can be subjects of different analyses.) Traditional pre-natal karyotyping only after in vitro culture of the cells. Amniocentesis earliest in week 16 of the pregnancy. Plus 2-3-4 weeks in culture. Too late. Prefer interphase cytogenetics. Chorionic villus sampling, sketch of CVS ►Can be performed on week 9-10.Delivers trophoblast cells (instead of embryonic), they come from the same zygote, buthigher risk of diagnostic error.

40. In utero ultrasound investigation: determination of nuchal translucency During week 10-12 (other auth-ors prefer 11-13) of the pregnancy, if the indi-cated dis-tance >3 mm, that speaks for risk of trisomy 21, especially if the nasal bone cannot be seen. A vizsgála-tot a 10-12. (másutt: a 11-13.) terhességi héten vég-zik. Ha 3 mm- nél vasta-gabbat mérnek, az Down szindróma gyanúját alapozza meg, külö-nösen, ha az orrcsont hiányával társul. ▼ ▲

41. Distribution of some relevant biochemical markers Some se-lected combina-tions are informa-tive. Down syn-dromic pregnancies = red curve. MoM = mul-tiple of themedian.

42. Cross-trimester marker ratios in prenatal screening (2007) Mehod Detection rate %False-positive First trimesterrate % {NT, PAPP-A, free beta-hCG} 83,75,1 Second trimester {AFP, uE3, total beta-hCG, INH-A} 84,46,6 Cross trimester ratios {NT, PAPP-A, free beta-hCG} + {AFP, uE3, total beta-hCG, INH-A} 90,83,1 Cross trimester ratios (another survey) {NT, PAPP-A, free beta-hCG} + {AFP, uE3, total beta-hCG, INH-A} 90,23,9 Some comments. The results of these investigations may be indicative of other chromosomal anomalies as well as malformations, too. E.g. AFP is elevated in the case of an open neural tube (and also in the case of twins). Detection rate % = retrospective analysis.

43. CT = cross tri-mester.Integ-rated = NT result included.Serum integ-rated = serum values only. What can we learn from this figure?:(1) Screening without karyotyping does not give reliable diagnosis. (2) Results of second trimester included ► rather late arrival.

44. Cost per Down syndromic pregnancy diagnosed (includes NT) At 95% detection rate the cost can vary between GBP 16,500 and 31,400. (British data, 2007.) # # # Financing national health care If a bigger population is investi-gated, the cost of screening 100,000 pregnant women can vary between GBP 3,540,000 and 6,740,000, de-pending on the extent of biochemi-cal markers involved. (Average = GBP 51.4 per case.)

45. PÄTAU SYNDROME

46. Patau* syndrome: trisomy 13 Multiple developmental anomalies, very limited lifetime. Trisomy 13 newborns usually die before age of 12 months. * Remember: there is no umlaut in the English, thus no ä for Klaus Pätau of a German immigrant ancestry.

47. Trisomy 13, symptoms and signs

48. EDWARDS SYNDROME: trisomy 18 Multiple developmental anomalies, very limited lifetime. Trisomy 18 newborns usually die before age of 12 months.

49. Trisomy 18, symptoms and signs

50. NUMBERS of GENES IDENTIFIED ON HUMAN CHROMOSOMES by the HUMAN GENOME PROJECT (HGP),as by February, 2007* # 1 = 2782 2610 # 9 = 1148 1076 # 17 = 1469 1394 # 2 = 1888 1748 # 10 = 1106 983 # 18 = 432 368 # 3 = 1469 1381 # 11 = 1848 1692 # 19 = 1695 1592 # 4 = 1154 1024 # 12 = 1370 1268 # 20 = 737 710 # 5 = 1268 1190 # 13 = 551 496 # 21 = 352 337 # 6 = 1505 1394 # 14 = 1275 1173 # 22 = 742 701 # 7 = 1452 1378 # 15 = 945 906X = 1336 1141 # 8 = 984 927 # 16 = 1109 1032Y = 307 255 Σ human genes (in this table) = 28,924 Remember: a gene here = a given protein coding sequence of the DNA.Small characters: data from January, 2006.* The figures are the same in January, 2008.