Joseph

Schlammadinger

retired associate

professor

Lectures in March, 2013

(revised versions)

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.

CYTO-GENETICS

2013

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.)

(G0) G1

M S 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.)

Arrangement

of chromosomes

in an

interphase

nucleus.(Computer

reconstruction in false colours.)

MITOSIS (first half)

Metaphase MITOSIS (second half cytokinesis)

Metaphase chromosome as seen in the electron microscope

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

LIGHT MICROSCOPIC INVESTIGATION OF HUMAN METAPAHSE CHROMOSOMES

I. 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.

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)

SHAPES OF CHROMOSOMES, I A: 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

Shapes of chromosomes, II

(for instance in mouse)

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.

ARRANGED KARYOGRAMS

FEMALE MALE

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.)

LIGHT MICROSCOPIC INVESTIGATION OF HUMAN METAPAHSE CHROMOSOMES

II. 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 and11/ describe karyotype.

Q-bands and G bands on chromosomes, and (Paris) ideogram

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.

NUMERICAL ABERRATIONS OF HUMAN CHROMOSOMES

NUMERICAL ABERRATIONS,POLYPLOIDIES

triploidy (3n = 69) tetraploidy (4n = 92)

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.

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.

Human numerical autosome aberrations (anomalies),aneuploidies

(Euploidy: see earlier. “an-” = privative prefix. Aneuploid = non-euploid.The chromosome number is not exact multiple of n.)

CAUSES OF ANEUPLOIDIES

1) Nondisjunction. Homologous chromosomes or sister-chromatids fail to separate from each other. It results in hyper- or hypohaploid 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.

2) 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.

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).

MITOTIC NONDISJUNCTION

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

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

DOWN SYNDROME, I

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, andCri du chat syndrome (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.

Down syndrome, II

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.

Symptoms and signs in Down syndrome

Increased liability to leukemiaIncreased liability to infections

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.

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

Risk of Down syndr.

Risk of Down syndr.

Risk of Down syndr.

<25 1:1500 33 1:570 42 1:65

25 1:1350 34 1:470 43 1:50

26 1:1300 35 1:380 44 1:35

27 1:1200 36 1:310 45 1:30

28 1:1100 37 1:240 46 1:20

29 1:1000 38 1:190 47 1:15

30 1:910 39 1:150 48 1:11

31 1:800 40 1:110 49 1:8

32 1:680 41 1:85 50 1:6

Maternal age

Maternal age

Maternal age

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.

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.

◄ 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, but

higher risk of diagnostic error.

In utero ultrasound investigation: determination of nuchal translucency

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.

▼

▲

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.

Distribution of some relevant biochemical markers

MoM = mul-tiple of themedian.

Some se-lected combina-tions are informa-tive.

Down syn-dromic pregnancies = red curve.

Cross-trimester marker ratios in prenatal screening (2007)

Mehod Detection rate % False-positive

First trimester rate %

{NT, PAPP-A, free beta-hCG} 83,7 5,1

Second trimester

{AFP, uE3, total beta-hCG, INH-A} 84,4 6,6

Cross trimester ratios

{NT, PAPP-A, free beta-hCG} +

{AFP, uE3, total beta-hCG, INH-A} 90,8 3,1

Cross trimester ratios (another survey)

{NT, PAPP-A, free beta-hCG} +

{AFP, uE3, total beta-hCG, INH-A} 90,2 3,9

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.

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.

Cost per Down syndromic pregnancy diagnosed

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.)

(includes NT)

PÄTAU SYNDROME

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.

Trisomy 13, symptoms and signs

EDWARDS SYNDROME: trisomy 18

Multiple developmental anomalies, very limited lifetime.

Trisomy 18 newborns usually die before age of 12 months.

Trisomy 18, symptoms and signs

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 = 945 906 X = 1336 1141

# 8 = 984 927 # 16 = 1109 1032 Y = 307 255

Σ human genes (in this table) = 28,924Remember: 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.

SEXUAL DIMORPHISM IN HUMANS

Is there any relationship between the dimorphism of sex chromosomes and the sex determination and sex development (resulting in the well known human sexual dimorphism)? – Yes.

Is it possible to compensate for the gene dosis differences (XX vs. XY) of females and males in the phenotype? (X = 1336, Y = 307 genes.) – Yes. See M. Lyon’s hypothesis of X inactivation.

SEX DETERMINATION IN HUMANSa genetic / chromosomal approach

Sexual development and some aspectsof its determination

1) Chromosomal sex XX vs. XY karyotype, SRY gene on the short arm of Y.

2) Gonadal sex Testosterone (androgene hormone) production by the fetal testes. Hormone receptor for testosterone encoded in the X chromosome (female development is automatic, this is suppressed in the case of the presence and action of Y chromosome and testosterone).

3) Genital sex Legal sex (as registered at birth).

4) Secondary sexual characteristics (developing mainly after puberty).

5) Psychosomatic sex (also determined by environmental effects).

Fate of the indifferent gonadal anlage

In the presence of Y chromosome, and es-pecially by the activity of SRY gene in it = development of testes.

In the absence of SRY effect, the ovary development is automatic.

Important early steps in sexual differentiation

(see precise details in embryology)

MALE: XY, SRY (sex determining region of the Y chromosome) activity. ► Indifferent genital ridge ► testes ► testosterone. ► Wolffian duct further develops (gives rise to prostate, ductus epidydimidis, vesicula seminalis etc.) ► while Müllerian duct disappears. ► Sinus urogenitalis closes. ► Tuberculum genitale develops into penis. ► Genital ridges close to form

scrotum.

FEMALE: XX automatism. No SRY effect, ► no testes, ► no testosterone effect, ► no development of Wolffian duct. ► Müllerian duct further develops and gives rise to tuba uterina, uterus, and vagina. ► Sinus urogenitalis remains open (labia minora, vestibulum vaginae). ► Tuber-culum genitale develops into clitoris. ► Genital ridges do not close, form labia maiora.

Genes on the Y and X chromosomes

YPseudoautosomal region (PSAR)*.SRY = sex determining region of the Y chromosome (previously: TDF =

testis determining factor, this term is still in use today).Localization: Yp11.3.ZFY = zinc finger protein Y (Yp11.32), a transcription regulator**.

XPseudoautosomal region (PSAR)*.TFM = gene of the androgene (dihydrotestosterone) receptor.ZFX = zinc finger protein X (Xp22.3-p21), a transcription regulator,and so on…99 other genes (including **), which code for proteins expressed in the

testes (or in tumors).

* PSAR on the short arm. Those in the Y and X are homologs to each other. (There are some pseudoautosomal genes in other locations, too.)

** There is a number of other transcription regulators.

SRY on the Y chromosome TFM on the X chromosome

The gene codes for a transription regulator protein. Mutation ►46,XY female.Translocation ► 46,XX male (Klinefelter syndrome).

Gene for dihydrotestosterone receptor (Xq11-q12). ║TFM = testicular feminisation, ║

Other name: AIS = androgen insensitivity syndrome. ║Complete form: Morris syndrome, ║

incomplete form: Reifenstein syndrome. ║Karyotype: 46,XY. Testes in the abdominal cavity. ║

No ovaries, no uterus. ║

Genes on autosomes

AMH, gene coding for anti-mullerian hormone. (Other name: Mullerian

Inhibiting Factor, MIF, or MIS) Responsible for the physiological de-generation of the Müller duct (which gives rise to internal female organs).Location: 19p13.3-p13.2. Expression: the encoded protein (560 amino acids) is produced in the testis, in the Sertoli cells. (In the same gonad the Leydig cells produce the testosterone, which is involved in the differentiation of the Wolff duct into prostate, seminal vesicle, spermatic duct, respectively. The Amh gene becomes silent after puberty.

AMHR = gene coding for the receptor of AMH (MIF) protein.

PMDS = persistent Mullerian duct syndrome. (For instance uterus in male, etc.)

Type I: defective Amh.Type II: defective Amhr.

GENE DOSAGE COMPENSATION

Although there is a number of characteristic phenotypic differences, there is no phenotypic sign of the more genes in females than in males.

Hypothesis of Mary Lyon: one out of the two X xchromosomes becomes inactive. (It forms a heterochromatic Barr-body attached to the internal surface of the nuclear envelope. If there are more than two X chromosomes, one remains active and all the others become inactivated: dosage compensation.)

X-inactivation is random, takes place in the 3rd embyonic week. If an X chromosome–by chance, either of paternal or maternal origin–, has been inactivated in a cell, in all descendants of that very cell the same X will be inactivated and forms Barr-body. This can be expressed as “inactivation is imprinted”.

Because of that random inactivation females are functional mosaics. Normally there is no preference which X to remain active. We have quite

a big number of data indicating the validity of the random type inactivation resulting in a closely fifty – fifty ratio. This is, however, a statistical value, which is a subject of considerable deviations, too.

One, two, and four Barr bodies, respectively, in nuclei of buccal epithelial cells. Barr body in polymorphonuclear

granulocyte nucleus (drumstick)

SEX CHROMOSOMAL ANEUPLOIDIESin HUMANS

KARYOTYPES and CONSEQUENCES

X MONOSOMY

Turner syndrome: 45,X.No Barr body.

Leading symptoms:

Sexual infantilism = immaturity of secondray sexual characteristics.Short stature.Cubitus valgus, pterygium colli, and so on.

Sterility (infertility) = No maturation and production of egg cells. Instead of ovarian tissue so called gonadal streak, which is made of connective tissue. Not only germ cells are not produced, neither estrogen, nor progesterone hormones. Consequently the level of gonadotropin is high.

Main symptoms and signs of Turner syndrome

Turner syndromepterygium colli gonadal streak

and its histology

No Grafian follicles, no meiotic divisions are seen in the histological picture.

No maturation of egg cells. No production of ovarial hormones.

Turner syndromelymphatic edema on the neck and on the feet

Karyotype: 45,X

Symptoms of Turner syndrome can be partially prevented

Early diagnosis (verified in the neonatal period), including immediate chromosome analysis (e.g. already from cord blood).

Hormonal treatment: (i) growth hormone, (ii) estorgen and progesterone.

Psychical/psychological support to the parents as well as to the Turner syndromic patient herself.

X chromosomal hyperdiploidies

47,XXXtrisomy X

49,XXXXXX pentasomy

X chromosomal hyperdiploidies

47,XXX (trisomy X) is not considered a syndrome: there are no characteristic signs and symptoms of this aneuploidy, the phe-notype is–in the majority of cases–normal*. It can result, how-ever, in the birth of a 47,XXY (Klinefelter syndromic) baby.(Theoretically the chance would be 1/4; 1/4 XX, 1/4 XXX, 1/4 XY, and 1/4 XXY. In fact the real risk is much lower.) In the case of triplo-X, prenatal chromosome analysis is strongly rec-ommended.

X tetrasomy (48,XXXX) is characterized by mental retardation;X pentasomy (49,XXXXX) patients show severe mental and corporal retardation in all cases, although in these aneuploidies only one X remains active, all others form Barr bodies.

* See table of spontaneous abortions and chromosome anomalies.

Sex chromosomal hyperdiploidies

Klinefelter sydrome: sex chromosome trisomy = 47,XXY.

Barr body is present in cell nuclei.

Leading symptoms: eunuchoid type tall stature, gynecomastia, no body hairs or very sparse, no beard, small testes with no Leydig and Sertoli cell functions.

Sterility (infertility): no spermiogenesis.

High gonadotrope hormone level in blood.

Klinefelter syndrome (47,XXY)

Symptoms and signs in Klinefelter syndrome

Klinefelter syndrome variants (48,XXXY, 49,XXXXY, 48,XXYY)

In the case of 47,XXY Klinefelter syndrome

mental abilities are of average in majority of patients.

If more than one extra X chromosome is present in the karyotype

(48,XXXY, 49,XXXXY) mental retardation or even severe mental

retardation is one of the leading symptoms, although only one X

chromosome remains active, all the others form Barr body.

47,XYY

No symptoms, therefore this is not a syndrome.

Risk: 47,XXY karyotype offspring.

Prenatal diagnosis stongly recommended.

STRUCTURAL ABERRATIONS OF HUMAN CHROMOSOMES

One should not forget the fact:there are a number of genes in one–light microscopically visible–band. Any deletion, duplication or rearrangement involves minimum 50 genes. (In high

resolution banding approximately 25.)

AUTOMATED INVESTIGATION OF HUMAN METAPAHSE CHROMOSOMES

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). 7/ Conventional light or fluorescent microscope. TV/CCD camera on its

exit port. 8/ Images to computer which has been programmed for automatic

chromosome analysis. Arranged karyogram on the monitor. 9/ The operator is allowed to correct the arrangement offered by the

computer.10/ The result is an ordered (arranged) karyogram. Evaluate and11/ establish karyotype. Save as a karyotype file.

STRUCTURAL CHROMOSOME ABERRATIONS

– DeletionRing chromosome formationMicrodeletion

– DuplicationMicroduplication (visible within the chromosome), microchromosome

– Isochromosome (deletion and duplication simultaneously)– Translocation

Unidirectional traslocationReciproc translocationRobertsonian translocation (centric fusion)

– InversionPericentricParacentric

– Break and gap– and some other forms (e.g. homogeneously staining region, double

minutes)

Deletionany chromosome fragment without a centromere will be lost

Ring chromosomethe two telomeric fragments are lost

Cri du chat syndrome (cat cry disease): 46,XX,5p–

Isochromosome formationInstead of the regular

separation of sister chromatids, the long and short arms, respectively, remain together, because of misdivision of the centromere. Consequently, in one daughter cell the short arm, in the other the long arm will be present in duplicated (trisomic) form, while the long arm and the short arm, respectively, will be monosomic in that very cell.

X long arm isochromosome has been described in Turner syndrome. Do not miss the point: that is X short arm monosomy at the same time.

Robertsonian translocation = centric fusion:46,XX,–14,t(14;21), Down syndrome

The Robertsonian translocation = centric fusion…

…may be the result of a new chromosomal abnormality = de novo mutation, but also can be inherited from a balanced carrier parent:45,XX,–14,–21,t(14;21). ►

Very important. A careful chromosomal diagnosis has to be established in any case of Down syndromic birth, and if translocation is discovered, the parents have to be investigated, too. Eventually some other balanced carrier(s) might be present in the family of the affected.

Theoretical gametic combinations in balanced t(14;21) translocation carriers

The theoretical risk in a family where one parent is a balanced carrier of a Robertsonian 14/21 translocation is calculated as follows:

1/3 normal,

1/3 balanced translocation carrier, and

1/3 unbalanced translocation, i.e. Down syndrome.

The empirical statistical risk is, however, somewhat better:

1/10 for Down syndrome if the mother, and

1/20 if the father is balanced carrier.

Even if we calculate with this latter figures, that is high enough to indicate: a prenatal chromosome diagnostics is a must. “Must” means: anyone in the health profession must be aware of the enhanced risk (like in the case of a pregnant woman ≥35), and is obliged to inform the parents of that risk. It is the parents’ decision, however, whether they accept the information obtained at the genetic counseling or not. Please note: no suggestion, no persuasion, no obligation, just information.

Other types of Robertsonian translocationst 21/22 Down syndrome Pätau syndrome

46,XX,–14,t(13;14) 45,XY,–13,–14,t(13,14)Pätau syndrome balaced carrier parent

Reciprocal (non Robertsonian) translocation

Enhanced risk for incorrect separation after meio-tic pairing, resulting in severe gametic (and zygotic) chromoso-mal aber-rations. (See also consider-ations on inversions below.)

Inversions

Like in the case of balanced translocations, inversions do not necessarily involve gene loss or gene duplication, i.e. the visible phenotype can be normal, without any indication of the presence of some chromosomal aberration in a balanced carrier.

If, however, during meiosis of a balanced carrier recombination(s) cause severe rearrangements, these latter may result in an unbalanced genome in the offspring, which at one end may be incompatible with life (e.g. infertility, or serial spontaneous abortions), on the other end may result in serious malformations.

Consequences of inversion and recombination in meiosis…

… and gamete formation after crossing over

and so on...

Familiar mental retardation (FMR)…

… fragile X (fraX, FRAXA) syndrome

1) Not a real break on the X chromosome, it can be evoked and made visible by certain cell culture technique (inhibition of DNA synthesis).

2) This abnormality can be made responsible for the fact, that mental retardation is more frequent among males than females (hemizygosity), although also heterozygous females may express the symptoms. The fragile site at Xq27.3 is associated with the fmr gene, i.e. patients with fragile X syndrome do not express the FMR1 gene.

3) More details in chapter on dynamic (expansive) mutations.

Fragile-X and familial mental retardation

▲

CHROMOSOMAL ABNORMALITIES and SPONTANEOUS ABORTIONS (in 100,000 pregnancies)

Karyotype Sp. abortion Livebirth Karyotype Sp. abortion Livebirth

Normal 7,500 84,450 47,XYY 4 46Trisomy 1 0 0 47,XXY 4 44 2 159 0 45,X 1,350 8 3 53 0 47,XXX 21 44 4 95 0 5 0 0 Translocations, 6 - 12 561 0 balanced 14 164 13 128 17 unbalanced 225 52 14 275 0 15 318 0 Triploidy 1,275 0 16 1,229 0 Tetraploidy 450 0 17 10 0 18 223 13 Others 280 49 19 - 20 52 0 21 350 11 3 Σ chromosomal 22 424 0 aberrations 7,500 550

–(13, 18 and 21) 3,176 0 Altogether 15,000 85,000

SISTER CHROMATID EXCHANGE (SCE)

Up to a limited number (approximately 5-10/metaphase) this phenomenon is considered normal. In certain hereditary diseases the frequency is, however, elevated, and that is indicative of the presence of e.g. Bloom syndrome, Fanconi anemia, xeroderma pigmentosum (all these are one or other kind of DNA repair defects).

SCE frequency is increased e.g. in radiation injury as well as in the presence of some chemical mutagens.

CHROMOSOME ABERRATIONS and CANCER, I

Philadelphia chromosome in

CML (chronic myelogeneous leukemia).

Reciproc

translocation ►►►

A fusion of ABL (proto-oncogene) and BCR gene.

Chromosome aberrations and cancer, II

t(8;14) in B-cell lymphomaIGH = immunoglobulin heavy chain gene. MYC = protooncogene, which is

activated to oncogene because of translocation.

Advantages of the FISH technique in general, and in the detection of minor chromosome

abnormalities

FISH = fluorescent in situ hybridization.

You know a given DNA sequence (e.g., from the results of the HGP). Synthesize a complementary single standed oligo-nucleotid probe. Label the latter with a fluorochrome.

Separate the two strands of the original double helix (e.g. by moderate heating), in situ, in the chromatin material.

Add probe. Let it anneal (hybridize) to the complementary strand (only to that, nowhere else), e.g., by slow cooling.

There are chromosome-specific (region-specific, band specific, etc.) probes as well as gene specific probes, and the like.

UP-TO-DATE DEVELOPMENTS of CYTOGENETICS

1/ Dividing cells are not inevitably necessary, 2/ interphase cell nuclei can also be investigated in some cases. 3/ Fluorescent in situ hybridization = FISH, with complementary single

stranded DNA probes (most commonly labeled oligonucleotides). 4/ These probes are: gene-specific, gene segment-specific, chromosome-

specific, chromosome segment-specific and so on, 5/ i.e. extremely high resolution can be achieved. 6/ Probes are mostly labeled with a fluorochrome, and the preps are 7/ investigated in fluorescence or in laser confocal microscope. 8/ CCD / TV camera ► computer, programmed for karyotypic and metaphase chromosome analysis, or a number of other evaluations. 9/ The investigator has a chance to reconsider and correct the result.10/ The goal of the investigation can be karyotyping, analysis of individual

chromosomes. Even one gene can be identified. And so on.11/ Another objective: CGH (comparative genomic hybridization) with

quantitative evaluation.

FURTHER DEVELOPMENTS in CYTOGENETICS

In situ PCR (polymerase chain reaction, amplification of a well defined, given sequence of a DNA molecule).

Qualitative and quantitative DNA analysis by PCR (with or without DNA sequenation),

with DNA chips, and many other recently developed methods.

For instance: one identifies a representative segment on the long arm of chromosome 21 at the DNA level and determines if it is present in 1× dose (normal), or in 1.5× doses (trisomy 21) in isolated DNA of the patient. Cheaper and faster method than traditional karyotyping.

Note, however. This diagnosis does not tell t(14;21) Robertsonian translocation Down syndrome from trisomy 21 “classical” Down syndrome. One has to investigate the presence or absence of 21p, too.

CHROMOSOME PAINTINGwith chromosome-specific fluorescent DNA probes

( 46,XY / 47,XY,+8 )

Trisomy 8 might be viable in the chromosome mosaic form, if only few cells are trisomic.

FISH, a way to the interphase cytogeneticsLeft: DiGeorge syndrome,del(22q11.21-q11.23), microdeletion.Right: Williams–Beuron syn-drome, del(7q11.2), microdeletion.

Detection of a marker (mar) microchromosome

MCB = multi color banding

UNIPARENTAL DISOMY (UPD)

Some conceptuses have a normal 46,XX or 46,XY karyotype, but have inherited two copies of the same chromosome from just one of the two parents. This may result in abnormal phenotypes which are different according to parental origin

of the relevant chromosome.

Isodisomy. Both homologs of the affected are identical.

Heterodisomy. Both homologs of one parent are present in the individual affected.

UPD is probably a result of an eventual correction of hyperdiploidy. The conceptus may be trisomic, but the zygote finds a way to get rid of the

supernumerary chromosome (in order to remain viable). For example:

15mat 1 + 15mat 2 + 15pat after correction 15mat 1 + l5pat, or 15mat 2 + 15pat, or 15mat 1 + 15mat 2 = UPD.

The first two versions will not be detected.

Prader–Willi and Angelman syndromes

46,XX or 46,XY individuals who inherit both copies of their chromosome #15 from their father, develop Angelman syndrome; if both copies of #15 are maternally inherited, Prader–Willi syndrome is seen.

Explanation. In non-UPD patients with Prader–Willi syndrome 15q12 is deleted from the paternal #15 chromosome, in Angelman syndrome the maternal #15 suffered a microdeletion of 15q12. If there is no paternal #15 in the karyotype, it means also the absence of the indicated region. This abnormality shows itself in the Prader–Willi syndrome.

The basic concept is found in the phenomenon of genomic imprinting. Some genes are active only on the maternal or only in the paternal chromosome. If the maternal region should be active, but that is missing, the situation is equivalent with the occurrence of a loss of function mutation. Prader–Willi and Angelam syndromes were identified originally as recessively transmitted human malformations.