Principles of Clinical Cytogenetics

1. Principles of Clinical Cytogenetics

2. 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 and array-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”

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. 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. Differential Coloring of All 24 Chromosome Types in Nuclei of Human Male Diploid Fibroblasts

15. 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.

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

17. Array-CGH analysis of DNA from peripheral blood of case 1 identifying a gain of distal 8q and confirming the loss of terminal 18p. In the test/reference fluorescence intensity ratio plot of the whole genome, the BAC, PAC and cosmid clones are sorted according to their genomic location along the chromosomes with the short arm on the left and the long arm on the right. Gaps were introduced to mark the border between two chromosomes. Other gaps correspond to heterochromatin. The profile shows a loss of the three most distal clones in subband 18p11.32 (below 7 SD from the mean) and a gain of 13 clones in subbands 8q24.23–q24.3 (above 7 SD from the mean).

18. Genome-wide results from a high resolution microarray. CNVs in the test genome relative to the reference genome are highlighted across all the chromosomes (duplications in green and deletions in red).

19. Karyotyping versus array-CGH Both karyotyping and arrays are genome-wide technologies which can be used to assess the presence of genomic imbalance such as CNVs. The primary difference between them is in the resolution. A standard G-banded karyotype usually has a resolution of around 5 Mb. Depending upon the particular array and how many DNA probes it uses, it is possible to detect changes greater than 1 Mb at low resolution or, changes as small as 10 kb at high resolution. Much smaller CNVs can be detected by using higher resolution technologies, which means that more pathogenic CNVs may be detected using modern arrays than through karyotyping.

20. However, because CNVs are relatively common throughout the genome, numerous benign CNVs will also be detected, so careful interpretation and follow-up testing is needed. This test measures gains and losses of copies of DNA. It will detect genomic imbalances such as aneuploidy, chromosomal deletions, or chromosomal duplications of the specific loci located on the array. It will not detect balanced rearrangements such as inversions, reciprocal translocations, Robertsonian translocations or insertions.

21. Chromosome Abnormalities • Numerical • Structural • Balanced • Unbalanced • Stable vs. unstable

22. 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)

23. Some Common Abbreviations

26. 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:

27. 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

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

29. 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

30. 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.

31. 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)

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

33. 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.

34. 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.

35. 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.

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

37. 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

38. 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.

39. Examples of structural rearrangements

40. 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).

41. 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

42. 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.

43. 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

44. 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.

45. 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

46. 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

47. A supernumerary ring marker originating from chromosome 16. The sample tested was amniotic fluid. Banded metaphase (left) and spectral karyotype (right) are shown. 

48. A supernumerary bisatellited marker originating from chromosome 22 detected in a peripheral blood sample from a 2-year-old boy. Banded metaphase (left) and spectral karyotype (right) are shown. Marker origin was confirmed by FISH using probes for 14/22 centromeres and the TUPLE1 gene locus (22q11.2)

49. 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 markers derived from centric portion of X. • Neocentromeres: contained in a subclass of marker chr.; small fragments of chr. arms that somehow acquired centromere activity

50. 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)