Principles of Clinical Cytogenetics

Clinical Cytogenetics is the study of chromosomes, their structure and their inheritance, as applied to the practice of medical genetics

Chromosome disorders form a major category of genetic disease:– Reproductive wastage, congenital malformation,

mental retardation (MR), and pathogenesis of cancer.

– Specific chr. abnormalities are responsible for 100’s of identifiable syndromes, collectively more common than single-gene disorders.

Prevalence of Cytogenetic DisordersPrevalence of Cytogenetic Disorders

~~ 1% of all live births1% of all live births

~ 2% of pregnancies in women older ~ 2% of pregnancies in women older than 35than 35

1/2 of all spontaneous first-trimester 1/2 of all spontaneous first-trimester abortionsabortions

Indications for a karyotypeIndications for a karyotype

Problems of early growth and development: failure to thrive, developmental delay, dysmorphic faces, multiple malformations, short stature, ambiguous genitalia and MR.

Stillbirth and neonatal death

Fertility problems: couples with a history of infertility or multiple pregnancy loss, women with amenorrhea

Family history: a known/suspected chr. abnormality in a first degree relative

Neoplasia

Pregnancy in a woman of advanced age (>35 yrs)

A uniform system of chr. classification is internationally accepted for identification.

The pattern of bands on each chr is numbered on each arm.

By this numbering, location of any band, DNA sequences and genes within it, and its involvement in a chr. abnormality can be precisely described

Chromosome IdentificationChromosome Identification

Culture (PB, fibroblasts, lymphobalstoid cell lines, Bone marrow, fetal cells)

Banding (G, Q, R)Special procedures (C-banding, high

resolution banding, fragile sites)Molecular cytogenetics (e.g., FISH, SKY,

CGH)

Ideogram, G-bands at metaphase, with about 400 bands/haploid karyotype.

At 550-band stage of condensation.

Metaphase, prometaphase and late prophase

Human chr’s are often classified by position of centromere into: metacentric, submetacentric, acrocentric.

Acrocentric (13,14,15,21,22) have small distinctive chromatin masses (satellites) attached to short arms by narrow stalks (secondary constrictions).– Stalks of these 5 pairs contain 100’s of copies of

genes encoding rRNA as well as a variety of repetitive sequences.

Fluorescence in situ Hybridization

Confluence of genomic and cytogenetic approaches=molecular cytogenetics

Examine presence or absence of a particular DNA sequence or evaluate the number or organization of a chr. or chromosomal region.

FISH at metaphase and interphase with 3 different types of probes. F-VIII, α-satellite (chr. 17), painting probe (chr. X)

FISH

2,3 and even 4-color applications to diagnose specific deletions, duplications, rearrangements in metaphase and interphase.

With specialized imaging procedure, 24 colors can be detected (SKY)

Chromosome and Genome Analysis by Use of Microarrays

Chromosome analysis can be performed at a genomic level by a variety of array-based methods using comparative genomic hybridization (CGH).

Assess the relative copy number of genomic DNA sequences in a comprehensive manner

Complements karyotyping and provides very sensitive, high resolution genome assessment.

Balanced translocations and rearrangements can not be resolved by array-based CGH.

Array CGH. Top: sample from a normal female. Bottom: sample from a male with trisomy 18.

Chromosome AbnormalitiesChromosome Abnormalities

NumericalStructural

– Balanced– Unbalanced– Stable vs. unstable

• The most common type of clinically significant chr. abnormality is aneuploidy. Always associated with physical or mental maldevelopment or both.

• Reciprocal translocations are also relatively common but usually have no phenotypic effect (risk of abnormal offspring)

• Chr. abnormalities are described by a standard set of abbreviations and nomenclature and technology used (e.g., FISH or microarray)

Some Common AbbreviationsSome Common AbbreviationsAbbrev. Meaning Example Condition

cen

del

der

dic

dup

fra

i

ins

inv

Centromere

Deletion

Derivative chr.

Dicentric chr.

Duplication

Fragile site

Isochromosome

Insertion

inversion

46,XX

46,XY

46,XX,del(5p)

der(1)

dic(X;Y)

46,Y,fra(X)(q27.3)

46,X,i(X)(q10)

inv(3)(p25q21)

Normal female karyotype

Normal male karyotype

Female with cri du chat s.

Translocation chr. Derived from chr.1 with cen of chr.1

Translocation chr. with centromeres of X and Y

Male with fragile X chr.

Female with isochr. Long arm X

Pericentric inv of chr.3

Abbrev. Meaning Example Condition

mar

mat

p

pat

q

r

rob

t

ter

Marker chr.

Maternal origin

Short arm

Paternal origin

Long arm

Ring

Robertsonian translocation

Translocation

Terminal or telomere

47,XX,+mar

47,XY,+der(1)mat

46,X,r(X)

Rob(13;21)(q10;q10)

46,XX,t(2;8)(q22;p21)

46,X,del(X)(pter q21:)

Female with an extra unidentified chr.

Male with an extra der(1) chr. Inherited from mother

Female with ring X chr.

Reunion at centromeric region of chr’s 13,21

Female with balanced translocation, breaks in 2q22 and 8p21

Deletion distal to q21 (i.e., q21 is present

Abbrev. Meaning Example Condition

+

-

:

::

/

ish

arr

cgh

Gain of

Loss of

break

Break & join

mosaicism

In situ hybridization

Array

Comparative genomic hybrid.

47,XX,+21

45,XX,-22

5qter 5p15:

2pter2q22::8p218pter

46,XX/47,XX,+8

ish 22q11.2(D22S75 X2)

46,XX.ish del(22)(q11.2)(D22S75-)

arr cgh 1-22(#BAC)x2, X(#BAC)x2, Y(#BAC)x0

arr cgh 1-22(#BAC)x2, X(#BAC)x1, Y(#BAC)x1

arr cgh 22q11.2(BAC name)x1

or

arr cgh 22q11.2(D22S75)x1

Female with trisomy 21

Female with monosomy 22

With deletion breakpoint in 5p15

Der(2) portion of t(2;8)

Probe for locus D22S75 in 22q11.2 (for DiGeorge S.). X2 = 2 signals (normal)

Female normal G-banding, deletion identified by FISH

Normal female array CGH pattern

Normal male array CGH

Loss of DiGeorge S. critical region

The phenotypic consequences of a chr. abnormality depend on its specific nature, resulting imbalance of involved genome parts, specific genes involved, and likelihood of its transmission.

A number of general principles that should be kept in mind:

Unbalanced karyotypes in liveborns: Unbalanced karyotypes in liveborns: General guidelines for counselingGeneral guidelines for counseling

Monosomies are more deleterious than trisomies Complete monosomies are generally not viable except for monosomy X Complete trisomies are viable for chr. 13,18,21,X,Y.Phenotype in partial aneusomies depends on: Size of unbalanced segment Imbalance monosomic or trisomic, and Region of genome and genes involvedIn a mosaic karyotype, “all bets are off”Rings give a phenotype specific to genome region involved, but are

commonly mosaic.Inversions Pericentric: risk of birth defects in offspring increases with size of

inversion Paracentric: very low risk of abnormal phenotype

Abnormalities of Chromosome Number

Heteroploid: any chromosome number other than 46

Euploid: exact multiple of haploidAneuploid: any chromosome number other

than euploid

Polyploidy- the presence of one or more extra complete sets of chromosomes in a cell

Remember that diploidy (2N) is normal for human somatic cells and haploidy (N) is normal for germ cells

Triploidy (3N) 69,XXX 69,XXY 69,XYY- Seen in fetuses, and lethal early in life - Observed in 1% to 3% of recognized conceptions- Usually caused by dispermy. Failure of one of the two meiotic divisions (diploid egg or sperm) may also occur.- Phenotype of triploid karyotype depends on source of extra chr. set:- Paternal abnormal placenta (partial hydatidiform moles),- Maternal spontaneously aborted earlier in pregnancy

Tetraploidy (4N) 92,XXXX 92,XXYY- Much rarer than triploidy - Seen in fetuses, and lethal early in life- Absence of XXXY or XYYY suggests failure of completion of an early cleavage division of zygote

AneuploidyAneuploidy

• Cells that do not contain a multiple of 23 chromosomes (n) – there are missing or extra chromosomes

• Most common and clinically significant chr disorder, present in at least 5% of all clinically recognized pregnancies

• Trisomy: presence of three copies of a chromosome. Monosomy (less often): presence of only one copy of a chromosome. Both can have severe phenotypic consequences.

• Most common type of trisomy in liveborns is trisomy 21. 47,XX or XY, +21: the constitution seen in 95% of Down syndrome.

• Other trisomies observed in liveborns include trisomy 13, 18.

• Notable that 13,18,21 are with low number of genes.• Monosomy for entire chr is almost always lethal. An

important exception is X (Turner syndrome).• Aneuploidy is generally caused by chromosome

nondisjunction• Premature separation of sister chromatids in M-I

instead of M-II (another mechanism)

Karyotype of fetal cells with trisomy 21 (Down syndrome)

Diagnosis: 47,XX, +21

Consequences of non-disjunction during meiosis I and II are different.

Non-disjunction has been associated with aberrations in frequency or placement, or both, of recombination events in meiosis-I. Too few (or even no) recombinations, or too close to centromere or telomere favor non-disjunction.

Classic nondisjunction: failure of chr’s either to pair or to recombine properly, or both.

Another mechanism involves premature separation of sister chromatids in meiosis I instead of II. Separated chromatids may by chance segregate to oocyte or to polar body unbalanced gamete.

More complicated forms of multiple aneuploidyMore complicated forms of multiple aneuploidy

A gamete has an extra representative of more than one chr.

Nondisjunction can occur at two successive meiotic divisions, or by chance in both male and female gametes

Extremely rare except for sex chr’s. Nondisjunction can occur in mitotic division after

zygote formation. If early clinically significant mosaicism. E.g., in some malignant cell lines and some cell cultures

Probes: yellow/white for chr. 18; red for X; green for Y.

Left: normal sperm cells

Middle: 24,XX sperm

Right: 24,XY sperm

An important diagnostic tool, especially prenatally, interphase multicolor FISH to evaluate 13,18,21,X,Y aneuploidy.

Multicolor FISH analysis of interphase amniotic fluid cells

Structural Abnormalities

• Breakage and reconstitution in an abnormal combination• Less common than aneuploidy• Present in about 1/375 newborns.• Chr rearrangements can occur spontaneously at low freq. &

may be induced by clastogens, e.g., IR, some viral infections, and many chemicals.

• Like numerical abnormalities may be present in all cells or in mosaic form

• Balanced - no net gain/loss of chromosomal material Unbalanced - gain/loss of chromosomal material Stable: passing through meiotic & mitotic divisions

unaltered– To be stable, a rearranged chr must have a functional

centromere and two telomeres.

Examples of structural rearrangements

Unbalanced Rearrangements

– The phenotype is likely abnormal. Any change that disturbs normal balance of functional genes abnormal development.

Deletions: lead to partial monosomy Duplications: lead to partial trisomy

– Large deletions/duplications (at least a few million bp) detected by karyotyping.

– Smaller deletions/duplications requires FISH or microarray CGH analysis.

Two-color FISH of a case with DiGeorge syndrome (deletion of 22q11.2).

Array CGH.

A: partial duplication of 12p in a patient with an apparently normal routine karyotype and symptoms of Pallister-Killian syndrome.

B: terminal deletion in 1p in a patient with mental retardation.

C: de novo deletion in 7q22 in a patient with a complex abnormal phenotype

An important class involves submicroscopic changes of a telomere region in patients with idiopathic MR. small deletions, duplications & translocations have been detected in several percent of such patients

Targeted cytogenetic or genomic analysis of telomeric and subtelomeric regions by FISH or array CGH may be indicated in unexplained MR (important for counseling)

A cryptic translocation in a developmentally delayed proband. Probes for telomere of chr. 3p (red) & chr. 11q (green). An unbalanced translocation b/w 3p and 11q.

Deletions

• Caused by a break in a chromosome with a resultant loss of acentric segment/ unequal crossing over/ abnormal segregation from a balanced (translocation/inversion) abnormality

• A carrier is monosomic for lost segment

• Haploinsufficiency for those lost genes

• Deletion may be terminal or interstitial

• Clinical consequences depend upon size of deleted segment and number and function of lost genes

Analysis:– large deletions – visible cytogenetic changes (~ 1

in 7000 live births)– small deletions – high resolution banding/

Southern Blot/ Exon specific PCR/ FISH with targeted probes/ array CGH

Numerous deletions have been identified in dysmorphic patients & prenatal diagnosis. Knowledge of functional genes lost & their relation to phenotype.

Duplications Originate by unequal crossing over or by abnormal

segregation from meiosis in a carrier of a translocation or inversion.

Often lead to some phenotypic abnormalities E.g., duplication of all or a portion of 12p leads to

Pallister-Killian syndrome: characteristic craniofacial features, MR, and other birth defects likely related to trisomy or tetrasomy for specific genes in duplicated region

Marker and Ring Chromosomes

Marker chromosomes: very small, unidentified chromosomes, frequently mosaic.

Usually extra to the normal chr. complement; Supernumerary chromosomes or Extra Structurally Abnormal Chr. (ESACs)

Tiny marker chromosomes consist of little more than centromeric heterochromatin

Precise identification requires various FISH probes (SKY)

Larger markers contain some material from one/both arms imbalance for genes present

Prenatal frequency of de novo marker chr. ~1/2500

Risk of fetal abnormality can range from very low to as high as 100% depending on marker origin

A relatively high proportion results from chr.15 and from sex chr’s. specific syndromes are associated with bisatellited chr.15 derived markers and with centric portion of X.

Neocentromeres: contained in a subclass of marker chr.; small fragments of chr. arms that somehow acquired centromere activity

• Marker chr. that lack telomeric Marker chr. that lack telomeric sequencessequences• Deletion occurs at both tips of a chr. Deletion occurs at both tips of a chr. followed by a joining of the “sticky” followed by a joining of the “sticky” chromosome endschromosome ends• Rare, but have been detected for every Rare, but have been detected for every chr.chr.• Mitotically stable if ring contains Mitotically stable if ring contains centromerecentromere• Problems during disjunction at Problems during disjunction at anaphaseanaphase 46,X,r(X)46,X,r(X)

Ring Chromosomes

Isochromosomes• One arm is missing & the other is One arm is missing & the other is

duplicated in a mirror-image duplicated in a mirror-image

• Consider a person with 46 chr. Consider a person with 46 chr. carrying an isochromosome and a carrying an isochromosome and a person with two normal homologs in person with two normal homologs in addition to the isochr. What are the addition to the isochr. What are the consequences?consequences?

At least two mechanisms:At least two mechanisms:

(1)(1) Misdivision through centromere in Misdivision through centromere in meiosis II, and more commonly:meiosis II, and more commonly:

(2)(2) Exchange involving one arm of a chr Exchange involving one arm of a chr and its homolog (or sister and its homolog (or sister chromatids). chromatids).

Isochromosomes cont..

The most common isochr. is i(Xq) in some individuals with Turner syndrome.

i(18p) and i(12p) have also been seenIsochr. are frequently seen in karyotypes of

solid and hematological malignancies

Dicentric Chromosomes

A rare type, in which two chr segments (from different chr or from two chromatids of a single one), each with a centromere, fuse end to end, with loss of acentric fragments.

May be mitotically stable, if one centromere is inactivated or if the 2 centromeres coordinate their movement at anaphase

Most commonly, involve the sex chr. or the acrocentric chr. (Robertsonian translocation).

Balanced RearrangementsBalanced Rearrangements

Do not usually have a phenotypic effect. All chr. material is present but packaged differently.

Important to distinguish truly balanced at molecular level.

Because of high frequency of copy number polymorphisms (differences of many million bps b/w unrelated individuals), concept of balanced/unbalanced is arbitrary

Even truly balanced can pose a threat to subsequent generation. Carriers are likely to produce a high freq. of unbalanced gametes, increased risk of having abnormal offspring. Risk can range from 1 to 20% depending on rearrangement.

There is a possibility that chr. break(s) will disrupt gene(s). E.g., X-linked disease in female carriers of balanced X;autosome translocations. Such instances useful to mapping gene(s) responsible for disease.

Inversions

Inversions that Inversions that involve the involve the centromere centromere = pericentric= pericentric

Inversions that do Inversions that do not involve the not involve the centromere centromere = paracentric= paracentric

46, XY, inv(10)(q11.23q26.3)46, XY, inv(10)(q11.23q26.3)

Caused by 2 breaks on a chromosome with inversion Caused by 2 breaks on a chromosome with inversion of the segment and reinsertion at the original siteof the segment and reinsertion at the original site

Inversions

• Pericentric inversions are easier to identify cytogenetically. • An inversion does not usually cause an abnormal phenotype in carriers. A carrier is at risk to produce unbalanced offspring due to challenges in pairing at meiosis.• Although recombination is somewhat suppressed within inversion loops, when it occurs it can unbalanced gametes.

When paracentric: the risk that a carrier will have a liveborn child with an abnormal karyotype is very low (acentric or dicentric may not lead to viable offspring)

Pericentric: can lead to gametes with duplications and deficiencies. Risk of a carrier producing an unbalanced karyotype is ~ 5% to 10% (but each inv. is associated with a particular risk). Large pericentric inv’s more likely to lead to viable recombinant offspring because unbalanced segments in recombinants are smaller.

TranslocationsTranslocations

Involve exchange of chr. segments b/w two, usually nonhomologous, chr’s.

two main types: reciprocal & Robertsonian.Reciprocal Translocations: Reciprocal exchange of broken-off fragments.

Usually only 2 chr’s involved (rare complex translocations involve 3 or more).

Relatively common, found in ~ 1/600 newborns. Usually harmless, although are more common in institutionalized MR individuals.

Associated with a high risk of unbalanced gametes and abnormal progeny.

Commonly found in couples with 2 or more spont. abortions and in infertile males.

Reciprocal Translocations

Reciprocal exchange of chromosomal Reciprocal exchange of chromosomal material between nonhomologous material between nonhomologous chromosomeschromosomes

46,XY,t(3;21)(q31;q21)

46,XY,t(1;7)(q43;p15)

Reciprocal Translocation & Meiosis

• When chr’s of a carrier pair at meiosis, a quadrivalent When chr’s of a carrier pair at meiosis, a quadrivalent figure is formed.figure is formed.• At anaphase, chr’s segregate in one of three ways: At anaphase, chr’s segregate in one of three ways: alternate, adjacent-1, and adjacent-2. Alternate, the usual alternate, adjacent-1, and adjacent-2. Alternate, the usual type of meiotic segregation type of meiotic segregation both types of gamete both types of gamete balanced.balanced.• In adjacent-1, In adjacent-1, homologous centromereshomologous centromeres go to go to separate separate daughter cellsdaughter cells, whereas in adjacent-2 (which , whereas in adjacent-2 (which is rareis rare), ), homologous centromeres pass to same daughter cell.homologous centromeres pass to same daughter cell.• Additionally, balanced translocation chr’s can also Additionally, balanced translocation chr’s can also segregate segregate 3:13:1, leading to gametes with 22 or 24 chr’s. Such , leading to gametes with 22 or 24 chr’s. Such 3:1 segregation is observed in 3:1 segregation is observed in 5-20% of sperm5-20% of sperm from from balanced translocation carrier, depending on specific balanced translocation carrier, depending on specific translocation.translocation.

Robertsonian Translocations

• Fusion of the long arms of 2 acrocentric Fusion of the long arms of 2 acrocentric chromosomes at the centromeres with loss chromosomes at the centromeres with loss of both short armsof both short arms• Involves chromosomes 13,14,15,21,22Involves chromosomes 13,14,15,21,22• carriers have 45 chr’s.carriers have 45 chr’s.• Rob translocation chr can be monocentric Rob translocation chr can be monocentric or pseudodicentric depending on or pseudodicentric depending on breakpoint locationbreakpoint location

45,XX, rob(14;21)(q10;q10)45,XX, rob(14;21)(q10;q10)

-13q14q and 14q21q are relatively common.

- 13q14q is found in ~ 1/1300, a common chr rearrangement in human.

- rare homozygotes for 13q14q exist. Phenotypically normal with 44 chr’s (no normal 13’s or 14’s).

- Carrier normal, risk of unbalanced gamete and thus offspring. Risk varies according to particular translocation and sex of carrier; carrier females have a higher risk of transmitting translocation to an affected child.

Robertsonian TranslocationsRobertsonian Translocations

2121

212114141414

Insertions

A non-reciprocal type of translocation. A segment removed from one chr and inserted into a different chr. in usual or inverted orientation.

Rare, as they require 3 breaks Abnormal segregation in an insertion carrier can

produce offspring with deletion or duplication as well as normal and balanced carriers.

Av. risk of producing an abnormal child is high, up to 50%, and prenatal diagnosis is indicated.

Mosaicism

Two or more different chr. complements present in an individual.

May be either numerical or less frequently structural Typically detected by conventional karyotyping but

can be suspected in interphase FISH or array CGH. A common cause is nondisjunction in an early

postzygotic mitotic division. e.g., 47,+21 zygote 46/47,+21 mosaic.

Effects on development vary with timing of nondisjunction event, nature of chr abnormality, proportions of different chr complements present, tissues affected.

An additional problem: proportions of different chr complements in tissue being analyzed.

Incidence of Chromosome AnomaliesIncidence of Chromosome Anomalies

The major numerical disorders are 3 autosomal trisomies (13,18,21), and 4 sex chr aneuploidies: Turner syndrome (usually, 45,X), Klinefilter syndrome (47,XXY), 47,XYY, and 47,XXX.

Triploidies and tetraploidies account for small % of cases, particularly in spontaneous abortions.

Live Births– Overall incidence ~ 1/160 births (0.7%)– Most autosomal abnormalities can be diagnosed at

birth, but most sex chr abnormalities (with exception of Turner) are not recognized clinically until puberty

– Balanced rearrangements are rarely identified clinically unless a carrier gives birth to a child with an unbalanced chr. complement and family studies initiated

– Unbalanced rearrangements are likely to come to clinical attention because of abnormal appearance and delayed physical and mental development

Parent-of-Origin EffectsParent-of-Origin Effects

Genomic Imprinting– For some disorders, expression of disease phenotypes depends on

parental origin of mutant allele or abnormal chr.– Differences in gene expression b/w allele inherited from mother

and allele inherited from father are the result of genomic imprinting.

– Imprinting is a normal process caused by alterations in chromatin that occur in germline of one parent, but not the other, at characteristic locations in the genome.

– The alterations include covalent modification of DNA, e.g., methylation of C 5-methyl cytosine, or modification or substitution in chromatin of specific histone types, which can influence gene expression.

– Imprinting affects expression but not DNA sequence. It is a reversible form of gene inactivation but not a mutation, i.e., epigenetic effect.

– Epigenetics are with significant influences on gene expression and phenotype both in normal individuals and a variety of disorders (cytogenetic, single gene and cancer)

– Imprinting takes place during gametogenesis, before fertilization, and marks certain genes as having come from mother or father.

– Imprint controls gene expression in some or all somatic tissues of embryo.

– Imprinted state persists postnatally into adulthood. Yet, imprinting must be reversible (e.g., during gametogenesis).

– Control over conversion appears to be governed by DNA elements called “imprinting centers” located within imprinted regions, their precise mechanism is unknown, they must initiate epigenetic change in chromatin, which then spreads outward over imprinted region.

Diagram of conversion of maternal and paternal imprinting during passage through germline to make male or female gametes. Erasure of imprint and conversion to that of other sex is marked by asterisk.

It is likely that several dozen or ~ 100 genes show imprinting effects.

Some regions contain a single imprinted gene; others contain clusters, spanning in some cases well over 1 Mb, of multiple imprinted genes

Only one allele (either maternal or paternal) is expressed. Non-imprinted loci (majority of loci) are expressed from both alleles.

Map of imprinted regions in human genome (gray: expressed only from maternal; blue: expressed from paternal)

Prader-Willi and Angelman SyndromesPrader-Willi and Angelman Syndromes PW is a relatively common dysmorphic syndrome: obesity,

excessive and indiscriminate eating habits, small hands short stature, hypogonadism, and mental retardation.

In ~ 70% of cases, there is a cytogenetic deletion involving (15q11-q13), occurring only on the chromosome 15 inherited from the patient's father

Thus, patients have genetic info in 15q11-q13 derived from their mothers.

In contrast, in ~ 70% of patients with the rare Angelman syndrome: unusual facial appearance, short stature, severe mental retardation, spasticity, and seizures, there is a deletion of ~ the same chr. region but on chr 15 inherited from mother.

Patients with AS, therefore, have genetic info. in 15q11-q13 derived from their fathers.

This demonstrates that parental origin of genetic material (15q11-q13) can have a profound effect on the clinical expression of a defect.

Prader-Willi syndrome. Left, Typical facies in a 9-year-old affected boy.

Right, Obesity, hypogonadism, and small hands and feet in a 9.5-year-old affected boy who also has short stature and developmental delay.

Two-color FISH analysis of proband with PWS, demonstrating deletion of 15q11-q13 on one homologue. Green signal is hybridization to α-satellite DNA at the centromere of chromosome 15. Red signal on distal 15q is a control single-copy probe. Red signal on proximal 15q is a probe for the SNRPN gene, which is present on one chromosome 15 (white arrow) but deleted from the other (dark arrow)

AS in a 4-year-old affected girl. Note wide stance and position of arms. Compare with phenotype of PWS.

Uniparental DisomyUniparental Disomy ~ 30% of PWS do not have cytogenetically detectable

deletions; instead, they have two normal chr. 15's, both inherited from the mother. i.e., uniparental disomy, defined as the presence of a disomic cell line containing two chromosomes, or portions thereof, inherited from only one parent.

If the identical chr. is present in duplicate, the situation is described as isodisomy; if both homologues from one parent are present, the situation is heterodisomy.

~ 3%-5% of AS also have uniparental disomy, two chr. 15's of paternal origin.

A few PWS and AS patients appear to have a defect in the imprinting center itself. As a result, the switch from female to male imprinting during spermatogenesis or from male to female imprinting during oogenesis.

Fertilization by a sperm carrying an abnormally persistent female imprint would produce a child with PWS; fertilization of an egg that bears an inappropriately persistent male imprint would result in AS.

E6-AP ubiquitinE6-AP ubiquitin--protein ligase geneprotein ligase gene

Finally, mutations in the maternal copy of a single gene, the E6-AP ubiquitin-protein ligase gene, have been found to cause AS.

E6-AP ubiquitin-protein ligase gene is located in 15q11-q13 and is normally imprinted (expressed only from the maternal allele) in the central nervous system.

It is likely that maternal 15q11-q13 deletions and the uniparental disomy of paternal 15 seen in AS cause the disorder because they result in loss of the maternal copy of this critically important, imprinted gene.

Mutations in a single imprinted gene have not yet been found in Prader-Willi syndrome.

  Prader-Willi Syndrome Angelman Syndrome

15q11-q13 deletion ~70% (paternal) ~70% (maternal)

Uniparental disomy ~30% (maternal) ~5% (paternal)

Single-gene mutation None detected E6-AP ubiquitin-protein ligase (10% of total but seen only in familial cases)

Imprinting center mutation

5% 5%

Unidentified <1% 10%-15%

Molecular Mechanisms Causing Prader-Willi and Angelman Syndromes

A few rare cystic fibrosis and short stature described with two identical copies of most or all of their maternal chromosome 7. In both cases, the mother happened to be a carrier for cystic fibrosis. The growth failure might be related to loss of unidentified paternally imprinted genes on chromosome 7.

Cytogenetics of Hydatidiform Moles and Cytogenetics of Hydatidiform Moles and

Ovarian TeratomasOvarian Teratomas

On occasion, in an abnormal pregnancy, the placenta is converted into a mass of tissue resembling a bunch of grapes, called a hydatid cyst.

This is due to abnormal growth of the chorionic villi, in which the epithelium proliferates and the stroma undergoes cystic cavitation. Such an abnormality is called a mole. A mole may be complete, with no fetus or normal placenta present, or partial, with remnants of placenta and perhaps a small atrophic fetus.

Hydatidiform mole (complete)

Most complete moles are diploid, 46,XX. The chr’s are all paternal in origin, however, and with

rare exceptions, all genetic loci are homozygous. Complete moles originate when a single 23,X sperm

fertilizes an ovum that lacks a nucleus, and its chromosomes then double.

The absence of any maternal contribution is thought to be responsible for the very abnormal development, with hyperplasia of the trophoblast and grossly disorganized or absent fetal tissue.

About half of all cases of choriocarcinoma (a malignant neoplasm of fetal, not maternal, tissue) develop from hydatidiform moles.

The reciprocal genetic condition is apparent in ovarian teratomas, benign tumors that arise from 46,XX cells containing only maternal chromosomes; no paternal contribution is evident.

Thus, normal fetal development requires both maternal and paternal genetic contributions. It appears that the paternal genome is especially important for extraembryonic development, whereas the maternal genome is critical for fetal development.

In contrast to complete moles, partial moles are triploid; in about two thirds of cases, the extra chromosome set is of paternal origin.

Comparing cases of maternal or paternal origin, fetal development is severely abnormal in both, but the defects are different. An extra paternal set results in abundant trophoblast but poor embryonic development, whereas an extra maternal set results in severe retardation of embryonic growth with a small, fibrotic placenta. The specificity of the effect is another example of genomic imprinting.

STUDIES OF CHROMOSOMES IN STUDIES OF CHROMOSOMES IN HUMAN MEIOSISHUMAN MEIOSIS

Two general approaches: 1) one can analyze abnormal meioses retrospectively, using DNA polymorphisms or cytogenetic heteromorphisms to study the parental origin of aneuploid fetuses or liveborns.

Extensive analysis of more than 1000 conceptuses has indicated a significantly different contribution of either maternal or paternal nondisjunction to different cytogenetic abnormalities

2) direct analysis of chromosomes in human germ cells. By use of FISH with chromosome-specific probes, a large number of sperm can be scored quickly to evaluate aneuploidy levels for individual human chromosomes.

A number of large studies have indicated chromosome-specific rates of disomy of about 1 in 1000 to 2000 sperm, with some variation between chromosomes. Nondisjunction of the sex chromosomes appears to be several-fold more frequent than nondisjunction of the autosomes.

Frequency of chromosomally abnormal sperm is elevated in males who exhibit infertility. This is an important area of investigation because of the increasing use of intracytoplasmic sperm injection (ICSI) in human in vitro fertilization (IVF) procedures; in many IVF centers, ICSI is the procedure of choice in male infertility cases.

There are a number of indications that suggest a sharp increase in chromosomal abnormalities (particularly involving the sex chromosomes) as well as imprinting defects in ICSI pregnancies.

Direct visualization of chromosomes during oogenesis is more difficult than during spermatogenesis.

As a result of improvements in IVF technology, however, oocytes can be obtained at the time of ovulation, matured in vitro, and examined by FISH, SKY, or array CGH during meiosis.

Such studies provide estimates of the frequency of nondisjunction in oogenesis as well as insights into mechanisms of maternal nondisjunction and the relationship between advancing maternal age, the frequency and placement of recombination events, and the increasing incidence of aneuploidy.

Combined immunohistochemical analysis of human oocyte chromosome bivalents. Each of the 23 bivalents is detected by an antibody to the synaptonemal complex (SCP3, in red). The location of the centromere in each bivalent is shown in blue with an antibody to centromere proteins (CREST). The position of recombination events (0 to 7 per bivalent in this cell) is indicated by presence of recombination protein (MLH1 in yellow).

MENDELIAN DISORDERS WITH MENDELIAN DISORDERS WITH

CYTOGENETIC EFFECTSCYTOGENETIC EFFECTS There are several rare single-gene syndromes, in

addition to the relatively common fragile X syndrome, in which there is a characteristic cytogenetic abnormality.

Collectively, these autosomal recessive disorders are referred to as chromosome instability syndromes. In each disorder, a detailed chromosome study can be an important element of diagnosis.

The nature of the chromosome defect and the underlying molecular defect in chromosome replication or repair is different in each of these disorders.

For example, Bloom syndrome is caused by a defect in a DNA

helicase that leads to a striking increase in somatic recombination and sister chromatid exchange.

Bloom syndrome is characterized by – Short stature – Immunodeficiency – Photosensitivity – Diabetes mellitus – Male sterility. – Chromosomal instability in Bloom syndrome results in a

high risk of cancer in affected individuals, that arise early in life.

ICF syndrome (characterized by immunodeficiency, centromeric instability, and facial anomalies) is caused by a deficiency in one of the DNA methyltransferases (DNMT3B gene) that are required for establishing and maintaining normal patterns of DNA methylation (at 5-methylcytosine residues) in the genome.

Chromosomes from patients with ICF syndrome show a characteristic abnormal association of pericentromeric heterochromatin involving chromosomes 1, 9, and 16.

Chromosomal abnormalities in metaphasic and interphasic cells of ICF patients: dual-color FISH was performed with chromosome 1 (green) and chromosome 16 (red) paint probes. Chromosomes and nuclei are counterstained with DAPI (blue). (a) and (c) show chromosomal abnormalities and micronuclei involving specifically chromosome 1, as frequently observed in the ICF1 and 1CF3 cell lines. (b) and (d) show chromosomal abnormalities and micronuclei involving both chromosomes 1 and 16, as frequently observed in the ICF2 cell line

Characteristic high frequency of sister chromatid exchanges in chromosomes from a patient with Bloom syndrome. Two exchanges are indicated by the arrows.

CYTOGENETIC ANALYSIS IN CANCERCYTOGENETIC ANALYSIS IN CANCER

An important area in cancer research is the delineation of cytogenetic changes in specific forms of cancer and the relation of the breakpoints of the various structural rearrangements to oncogenes.

The cytogenetic changes seen in cancer cells are numerous and diverse. Many are repeatedly seen in the same type of tumor.

Several hundred nonrandom chromosome changes involving all chromosomes except the Y chromosome have been identified in various neoplasias.

Characteristic Chromosome Translocations in Selected Human Malignant Neoplasms

Neoplasm Chromosome Translocation

Percentage of Cases

Proto-oncogene Affected

Burkitt lymphoma t(8;14)(q24;q32) 80%

t(8;22)(q24;q11) 15% MYC

t(2;8)(q11;q24) 5%

Chronic myelogenous leukemiat (9;22)(q34;q11) 90%-95% BCR-ABL

Acute lymphocytic leukemia t(9;22)(q34;q11) 10%-15% BCR-ABL

Acute lymphoblastic leukemia t(1;19)(q23;p13) 3%-6% TCF3-PBX1

Acute promyelocytic leukemia t(15;17)(q22;q11) ~95% RARA-PML

Chronic lymphocytic leukemia t(11;14)(q13;q32) 10%-30% BCL1

Follicular lymphoma t(14;18)(q32;q21) ~100% BCL2

The association of cytogenetic and genome analysis with tumor type and with the effectiveness of therapy is already an important part of the management of patients with cancer.

Their detection in clinical cytogenetics laboratories, by use of FISH, SKY, and array CGH, can have important diagnostic and prognostic value for oncologists.