1. Principles of Clinical Cytogenetics

2. Chromosome disorders form a major category of genetic disease:
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.

3. Prevalence of Cytogenetic Disorders
~ 1% of all live births
~ 2% of pregnancies in women older than 35
1/2 of all spontaneous first-trimester abortions

4. Indications 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)

5. 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

6. Chromosome 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)

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

8. At 550-band stage of condensation.

9. Metaphase, prometaphase and late prophase
400, 550, 800 bands levels
Metaphase, prometaphase and late prophase

10. 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.

11. 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.

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

13. 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)

14. 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.

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

16. Chromosome Abnormalities
Numerical
Structural
Balanced
Unbalanced
Stable vs. unstable

17. The most common type of clinically significant chr
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)

18. Some Common Abbreviations
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

19. 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

20. 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:
2pter2q22::8p218pter
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

21. The phenotypic consequences of a chr
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:

22. Unbalanced karyotypes in liveborns: General 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 involved
In 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

23. Abnormalities of Chromosome Number
Heteroploid: any chromosome number other than 46
Euploid: exact multiple of haploid
Aneuploid: any chromosome number other than euploid

24. Triploidy (3N) 69,XXX 69,XXY 69,XYY
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

25. Aneuploidy
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.

26. Most common type of trisomy in liveborns is trisomy 21
Most common type of trisomy in liveborns is trisomy ,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)

27. Karyotype of fetal cells with trisomy 21 (Down syndrome)
Diagnosis: 47,XX, +21

28. 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.

29. 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.

30. More 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

31. Probes: yellow/white for chr. 18; red for X; green for Y.
Left: normal sperm cells
Middle: 24,XX sperm
Right: 24,XY sperm

32. 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

33. 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.

34. Examples of structural rearrangements

35. 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).

36. B: terminal deletion in 1p in a patient with mental retardation.
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
characteristic Craniofacial features/MR/+ other birth defects

37. 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.

38. 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

39. 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.

40. 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

41. 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

42. 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

43. Ring Chromosomes
Marker chr. that lack telomeric sequences
Deletion occurs at both tips of a chr. followed by a joining of the “sticky” chromosome ends
Rare, but have been detected for every chr.
Mitotically stable if ring contains centromere
Problems during disjunction at anaphase
46,X,r(X)

44. Isochromosomes
One arm is missing & the other is duplicated in a mirror-image
Consider a person with 46 chr. carrying an isochromosome and a person with two normal homologs in addition to the isochr. What are the consequences?
At least two mechanisms:
Misdivision through centromere in meiosis II, and more commonly:
Exchange involving one arm of a chr and its homolog (or sister chromatids).

45. Isochromosomes cont..
The most common isochr. is i(Xq) in some individuals with Turner syndrome.
i(18p) and i(12p) have also been seen
Isochr. are frequently seen in karyotypes of solid and hematological malignancies

46. 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).

47. Balanced 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

48. Even truly balanced can pose a threat to subsequent generation
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.

49. Inversions
Caused by 2 breaks on a chromosome with inversion of the segment and reinsertion at the original site
Inversions that involve the centromere
= pericentric
Inversions that do not involve the centromere
= paracentric
46, XY, inv(10)(q11.23q26.3)

50. 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.

51. 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.

53. Translocations
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.

54. Reciprocal Translocations
Reciprocal exchange of chromosomal material between nonhomologous chromosomes
46,XY,t(3;21)(q31;q21)

55. 46,XY,t(1;7)(q43;p15)

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

58. Robertsonian Translocations
Fusion of the long arms of 2 acrocentric chromosomes at the centromeres with loss of both short arms
Involves chromosomes 13,14,15,21,22
carriers have 45 chr’s.
Rob translocation chr can be monocentric or pseudodicentric depending on breakpoint location
45,XX, rob(14;21)(q10;q10)

59. 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.

60. Robertsonian Translocations21 14

61. 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.

62. 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.

63. Incidence 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.

64. 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

65. Parent-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.

66. Imprinting affects expression but not DNA sequence
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.

67. 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.

68. 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)
Some regions contain clusters, some of which paternally imprinted and some maternally.

69. Prader-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.

70. Prader-Willi syndrome
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.

71. 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)

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

73. Uniparental 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.

74. 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.

75. E6-AP ubiquitin-protein 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.

76. Molecular Mechanisms Causing Prader-Willi and Angelman Syndromes
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%
Unidentified
<1%
10%-15%

77. 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.

78. Cytogenetics of Hydatidiform Moles and Ovarian 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.

79. Hydatidiform mole (complete)
Curettage specimen
Hydatidiform mole (complete)

80. 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.

81. 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.

82. 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.

83. STUDIES OF CHROMOSOMES IN HUMAN 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
for example, maternal nondisjunction accounts for more than 90% of cases of trisomy 21 and fully 100% of trisomy 16 but only about half of cases of Klinefelter syndrome (47,XXY) and only 20% to 30% of Turner syndrome (45,X).

84. 2) direct analysis of chromosomes in human germ cells
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.

85. 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.

86. 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.

87. 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).

88. MENDELIAN DISORDERS WITH CYTOGENETIC 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.

89. Bloom syndrome is characterized by
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.

90. 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.
ICF syndrome is a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability and facial anomalies (Ehrlich, 2003 ; Wijmenga et al., 2000 ). Individuals with this disease show combined immunodeficiency, including absence or severe reduction of at least two classes of immunoglobulin and a reduced number of T cells, making them prone to infections and death before adulthood. Centromeric instability is characterized by the formation of radiated chromosomes (chromosome 1, 9 and 16) due to demethylation at cytosine residues in classical satellites 2 and 3 at juxtacentromeric regions of these chromosomes. Facial anomalies include hypertelorism, flat nasal bridge, low set ears, protrusion of the tongue, epicanthal folds, micrognathia and high forehead. Most individuals also exhibit growth and mental retardation. Several studies have demonstrated that ICF syndrome is caused by homozygous or compound heterozygous mutations of the DNA methyltransferase 3B (DNMT3B) gene

91. 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

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

93. CYTOGENETIC 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.

94. Chromosome Translocation Percentage of Cases Proto-oncogene Affected
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%
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
PBX1 ia a transcription factor

95. 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.