1. A PPLIED C YTOGENETICS G ENETICS 202 Jon Bernstein Department of Pediatrics September 29 th, 2015

2. Learning Goals – Applied Cytogenetics Learn normal chromosome structure and nomenclature Gain familiarity with the results of errors in Meiosis and Mitosis, how they are diagnosed and how the behave in families ◦ Numerical chromosome abnormalities ◦ Isochromosomes ◦ Ring chromosomes ◦ Translocations  Reciprocal and Robertsonian translocations ◦ Inversions ◦ Chromosomal deletions and duplications  Microdeletions and microduplications (See short videos on coursework for additional detail)

3. Scales of Genetic Variation Structural Variant (SV) Small insertion or deletion (indel) Single nucleotide variant (SNV) Nature Reviews Genetics 12, 363-376 (May 2011) ACTGATCCG ACTGATAACCCG ACTGCG ACTGATCCG ACTGGTCCG

4. Learning Goals – Applied Cytogenetics Understand the methods, strengths and limitations of commonly used cytogenetic tests ◦ Karyotype ◦ FISH (Fluorescence in situ hybridization) ◦ Array CGH (comparative genomic hybridization) ◦ (See short videos on coursework for additional detail)

5. Applied Cytogenetics Outline for lecture ◦ The karyotype, normal chromosome structure ◦ Gallery of cytogenetic tests and anomalies ◦ Clinical cases demonstrating  Microscopic chromosome abnormalities  Seen on karyotype, Down syndrome, Turner syndrome, Inversion  Submicroscopic chromosome abnormalities  Seen by FISH or CGH, 22q11 deletion syndrome

6. Visualizing chromosomes and their types Cells to Tissues, Stanford Cytogenetics Lab MetacentricSubmetacentric Acrocentric Basepair #1 Basepair #247 Million #1 #14 #9

7. Karyotype Resolution High Resolution Karyotype, 550 bands or greater Standard Resolution Karyotype, 400 bands

8. Gallery of cytogenetic tests wikipedia.org Karyotype FISH – Fluorescence in situ hybridization DNA Microarray / Array Comparative Genomic Hybridization Test characteristics Resolution (size of variants detected) Ability to assess copy number (quantity of DNA) Ability to assess location (position of DNA)

9. Gallery of anomalies Trisomy

10. Interpreting cytogenetic test results What am I seeing? ◦ Is there a gain or loss of genetic material? Who has the change -- is it new or inherited?  If new, when and where could the change have occurred?  If inherited, did it malsegregate during meiosis?

11. Interpreting cytogenetic test results What does this result mean? ◦ Does this test result support a specific diagnosis? ◦ Can the genetic change be passed on by the patient? ◦ Are the patient’s parents or other relatives at risk of having a child with a similar genetic change?

12. Clinical Case #1 You have just examined an infant girl with low muscle tone and subtly distinctive facial features. A chromosome analysis is ordered and the following karyotype is seen: New Clinical Genetics 2e Andrew Read and Dian Donnai ISBN: 9781904842804 © Scion Publishing Ltd, 2011

13. Diagnostic Karyotype

14. Down Syndrome Occurs in 1/600 - 1/1000 births Due to extra #21 chromosome q arm ◦ ~95% of cases with trisomy 21, 47,XX +21 or 47,XY+21 ◦ Due to non–disjunction, typically in maternal meiosis I Diagnosis can be confirmed by chromosome analysis

15. Down Syndrome – Health Risks Newborn PeriodChildhoodAdulthood Congenital heart diseaseIntellectual disabilityDementia Duodenal atresiaHypothyroidismObesity Celiac diseaseMale infertility Dry skin

16. Recurrence risk counseling for Down syndrome For trisomy 21 the estimated recurrence risk is the greater of 1% or the maternal age related risk. A minority of cases of Down syndrome are due to a Robertsonian translocation and have a higher risk of familial recurrence.

17. Translocation involving acrocentric chromosomes ◦ 13, 14, 15, 21, 22 Breaks are at/near the centromere A centric fusion of q arms Acrocentric short arms contain redundant material Robertsonian Translocation Acrocentric

18. Robertsonian Translocation New Clinical Genetics 2e Andrew Read and Dian Donnai ISBN: 9781904842804 © Scion Publishing Ltd, 2011 45, XY, der(14;21)(q10;q10)

19. Robertsonian Translocation

20. Recurrence risk counseling for Down syndrome In translocation Down syndrome where the mother carries a Robertsonian translocation the risk of recurrence is estimated to be 10%. ◦ What if the translocation is a new occurrence? When a parent carries a 21;21 isochromosome the risk of recurrence is estimated to be 100%

21. Metacentric chromosome consisting of genetically identical arms with all loci equidistant from the centromere Believed to result from transverse rather than longitudinal division of the centromere in meiosis or mitosis Isochromosmes http://ghr.nlm.nih.gov/handbook/illustrations/isochromosomes

22. Clinical Case #2 Your are evaluating a young girl in clinic with short stature. As an infant she had “puffy feet.” A chromosome analysis is ordered and the following karyotype is seen: Stanford Medical Genetics http://learn.genetics.utah.edu/content/disorders/whataregd/turner/

23. Diagnostic karyotype

24. Turner Syndrome, 45,X Karyotype Birth prevalence 1:2500-1:5000 ◦ Represents 1-2% of all conceptuses with Turner syndrome; >90% result in pregnancy loss ◦ Reason for decreased viability incompletely understood Paternal X much more likely to be missing ◦ No maternal age effect ◦ Low recurrence risk

25. Turner syndrome Cardiac anomalies –Coarctation of the aorta –Bicuspid aortic valve Renal Anomalies –Horseshoe kidney Other –Congenital lymphedema –Osteoporosis Growth and development Short stature Normal intelligence Difficulties with spatial orientation and math

26. Other forms of Turner syndrome - mosaicism Mosaicism: The presence of two or more genetically different cell lines in an individual. ◦ Degree of mosaicism can vary from tissue to tissue ◦ The level of mosaicism in one tissue is not predictive of the level in others Mosaic Turner syndrome ◦ 45,X and another cell line - often 46,XX

27. Other forms of Turner syndrome – ring chromosome Formed by a double break, with the “broken ends” reuniting into a ring configuration Usually associated with some deletion of the chromosomal ends

28. Clinical Case #3 A 32 year old man is a carrier of a reciprocal translocation. What implications may this have for his offspring?

29. Balanced Reciprocal Translocation New Clinical Genetics 2e Andrew Read and Dian Donnai ISBN: 9781904842804 © Scion Publishing Ltd, 2011

30. Segregation of a reciprocal translocation

31. Reciprocal translocations Carriers of balanced reciprocal translocations typically have no clinical manifestations ◦ No net gain or loss of genetic information Reciprocal translocation carriers, like Robertsonian translocation carriers, are at risk to have offspring with unbalanced chromosome complements

32. Clinical Case #4 A newborn boy has a cleft palate and a short jaw. Amniocentesis karyotype (400 band resolution) was reported as normal.

33. New Clinical Genetics 2e Andrew Read and Dian Donnai ISBN: 9781904842804 © Scion Publishing Ltd, 2011 Array CGH

34. An array CGH study is performed and shows an 8 Mb duplication on 18p and a 7.5 Mb deletion on 18q.

35. Synthesis question Why might the amniocentesis karyotype have been reported as normal? ◦ An error? ◦ Differences in the pre and postnatal testing methods?

36. Possibly. A deletion and a duplication - two independent events?

37. Or, the result of meiotic segregation of a pericentric inversion. ◦ A chromosomal segment spanning the centromere is reversed in orientation ◦ Typically, no or minimal genetic material lost or gained in carrier

38. Chromosome Inversions

39. Segregation of pericentric inversions Modern Genetic Analysis. Griffiths AJF, Gelbart WM, Miller JH, et al. New York: W. H. Freeman; 1999.W. H. Freeman CopyrightCopyright © 1999, W. H. Freeman and Company. NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health. http://www.ncbi.nlm.nih.gov/books/NBK21367/ Stoler et al., N Engl J Med 2004;351:2319-26.

40. Fluorescence in situ Hybridization http://www.genome.gov/glossary/?id=65 Stanford Cytogenetics Lab – Athena Cherry wikipedia.org Interphase Metaphase

41. FISH study for case 4 Red=18p probe Green = 18q probe Patient Normal control Patient’s parent

42. Pericentric inversion carriers can produce gametes with duplication or deletion of chromosome ends ◦ Depending on the inversion, approximately 10% risk to have a child with an unbalanced karyotype Low risk for paracentric inversion carrier to have child with abnormal karytoype ◦ Recombinant chromosomes are acentric /dicentric and not viable Chromosome Inversions

43. Case #4 summary – Increased resolution ->able to detect smaller changes Normal amniocentesis karyotype ◦ Resolution only 400 bands 8p duplication and 8q deletion detected by aCGH and confirmed with position information by FISH ◦ Resolution is submicroscopic, smaller than visible bands ◦ These two events are the result of a parental pericentric inversion (balanced)

44. Clinical Case #5 Pre-school aged girl with a history of difficulty with articulation. Array CGH shows an interstitial deletion at chromosome 22q11.2 New Clinical Genetics 2e Andrew Read and Dian Donnai ISBN: 9781904842804 © Scion Publishing Ltd, 2011

45. 22q11.2 deletion syndrome Contiguous gene deletion syndrome resulting from interstitial microdeletion at 22q11.2 Clinical features ◦ Cardiac Anomalies ◦ Immune Dysfunction ◦ Craniofacial Anomalies ◦ Developmental Difficulties

46. Low Copy Repeats on 22q Deletions and duplications at 22q11 due to low copy repeats. Genome Res. 2007. 17: 451-460.

47. Synthesis question What are the chances that the parent of a child with a mosaic 22q11 deletion syndrome would carry the deletion? Mosaic microdeletion

48. Array CGH - Variants of Unknown Significance Is it inherited or de novo (new) Has it been seen before in other unrelated individuals ◦ The medical literature ◦ Online databases  Decipher http://decipher.sanger.ac.uk/http://decipher.sanger.ac.uk/

49. Lecture Summary Chromosome abnormalities observed under the light microscope include: ◦ Numerical abnormalities ◦ Robertsonian translocations ◦ Isochromosomes ◦ Ring chromosomes ◦ Inversions ◦ Reciprocal translocations The recurrence risk for a chromosome anomaly depends on the type of the anomaly and its origin

50. Summary There is a wide variety of submicroscopic variation in the human genome ◦ Microdeletions ◦ Microduplications  Pathogenic microduplications and microduplications can result in a variety of syndromes Copy number variants/Structural variants

51. Review Question An increased risk of recurrence in Down syndrome is potentially associated with the presence of: ◦ A) Mosaicism ◦ B) A Robertsonian translocation ◦ C) A ring chromosome ◦ D) A metacentric chromosome

52. Review Question Which type of translocation is more likely to result in a phenotype due to disruption of genetic material at a breakpoint? ◦ A) Robertsonian translocation ◦ B) Reciprocal translocation

53. Review Question You have performed array CGH on a patient with multiple congenital anomalies. Which cytogenetic abnormalities would be detectable by CGH? ◦ A) Robertsonian translocation ◦ B) Inversion ◦ C) Balanced reciprocal translocation ◦ D) Ring chromosome

54. Case for small group session Chief complaint (CC) ◦ You are asked to evaluate a 5 year old girl with a history of developmental delay currently hospitalized for poorly controlled seizures.

55. History of present illness (HPI) In addition to developmental delay and seizures, on exam the patient also has fleshy hands and thin toenails. She has a history of multiple urinary tract infections. The patient was born at term (40 weeks). Her mother's pregnancy was notable for febrile illness in the second trimester. An MRI of her head was normal.

56. Family History (FH) The patient has an older brother and a younger sister who are developing typically. Her parents have also had a pregnancy which ended in a miscarriage. The patient has a paternal cousin through her father’s sister with mild intellectual disability.