M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Learning Cycle Stage(s): Explore/Explain/Elaborate/Evaluate Adapted from: Adventures in Karyotyping Marcy Demsky, Sachem North High School NY Theresa Mercado, State University of New York at Stony Brook, NY ASHG Concept(s) Addressed: Nature of gene c material #2 Time Required: 130‐140 minutes Lesson 2: Analyzing Chromosomes [Adapted from h p://home.earthlink.net/~heinabilene/karyotypes/karyoty.htm] Explore 1) Have students read “Introduc on to Karyotyping” (Appendix I). Discuss the following ques ons with the class:  What is the purpose of a karyotype? What informa on does it tell you?  How is karyotyping done? Why is it important that cells are arrested at metaphase?  Since we are dealing with human karyotypes, how many matched pairs of chromosomes would you expect to see for a female? For a male? 2) For this ac vity, students will work as cytogene c technologists to complete a par al karyotype by cu ng out the remaining chromosomes in the spread and matching them with their homologs. Students should then determine the chromosome complement of their karyotype (e.g., 46, XX). Pass out copies of:  “Classifica on of Chromosomes for Karyotyping” (Appendix II),  the par ally completed karyotype (Appendix III),  and the corresponding metaphase spread (Appendix IV).  Going further: Students can explore the careers of various gene cs professionals using the fol‐
lowing resource: h p://www.ashg.org/educa on/careers.shtml  Prin ng of the karyotypes from the Appendixes provided should be done on a good quality printer for op mal chromosome visualiza on. Explain 1) Discuss the results of the karyotype as a class. Ask students what the chromosome complement of the pa ent is. Is the pa ent male or female? How do they know? Are any chromosomal abnor‐
mali es present?  Ask students if they no ce anything about the homologous pairs of chromosomes. Hopefully students will no ce that in this case, the homologs are iden cal. Ask if this is the case in real life. Let them know that this was done on purpose to get them accustomed to matching the chromosomes. In the next part of the lab, they will encounter a “real” metaphase spread and the homologs will be different. 2) Introduce students to the two broad categories of chromosome abnormali es, numerical abnor‐
mali es and structural abnormali es. Discuss how these types of abnormali es occur (students should already be familiar with meiosis) through non‐disjunc on and errant repair of double‐
stranded DNA breaks, respec vely. Introduce the terms monosomy, trisomy, and transloca on. Ask students how they think these abnormali es might affect a pa ent. 20 min 20‐30 min 5 min 15 min The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Elborate 1) Tell students that now that they have some experience as cytogene c techonologists, they will re‐
visit the case studies they built pedigrees for in the previous lesson. Give each group the appropri‐
ate parental karyotypes (Appendix V), an envelope of the appropriate pre‐cut fetal chromosomes (Appendix VI), double‐sided tape, and blank paper. Each group should create a fetal karyotype, and then determine the chromosomal complement of the pa ent, the pa ent’s gender, whether or not a chromosomal abnormality exists, and, if so, what that abnormality is. a. To prepare re‐usable pre‐cut chromosomes, discretely label the backs of each chromosome in Appendix VI with family name and chromosome number, laminate the sheets, then cut out the chromosomes and place each set in an envelope labeled with the family name. b. To help students correctly iden fy chromosomes, it is helpful to cut the chromosomes from copies of the fetal karyotype answer keys such that one homolog retains the chromosome number. c. Although they will not be directly used in this ac vity, students may also be given copies of the original metaphase spread (Appendix VII) to emphasize that some of the work has already been done. 2) Have each group prepare a short, five minute case review presenta on of their pedigree and kary‐
otype results for the class using the Case Review Prepara on Worksheet (Appendix VIII). Students can find more informa on about par cular abnormali es by visi ng h p://home.earthlink.net/
~heinabilene/karyotypes/karyoty.htm. 3) During the presenta ons, students from each group should take turns addressing the ques ons from the prepara on worksheet using their assembled pedigree and karyotypes for reference (use a document viewer to project them to the class). Have students in the audience keep track of each case using the Case Review Analysis Worksheet (Appendix IX). Evaluate Students will reprise their roles as gene c counselors and compose a le er to the parents of their pa ent explaining their findings. Give students a copy of both the le er guidelines (Appendix X) and the evalua on rubric (Appendix XI). While students may work in their groups to structure the le er, each student should individually submit a professional le er wri en in their own words. a. The group focusing on the Doherty case should be informed that while karyotyping revealed a normal chromosomal complement, subsequent screening revealed a genotype associated with cys c fibrosis. b. Likewise, the group focusing on the Jackson case should be informed that further screening indicated the presence of a genotype associated with sickle cell anemia. 20 min 20 min 30 min HW The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Appendix I CYTOGENETICS LAB ‐ KARYOTYPING Name:________________________________________________ Date:________________ Period:____________ Special thanks to tmercado@notes.cc.sunysb.edu, and nanhei@hawley.esc14.net Metaphase Spread Karyotype Introduc on to Karyotyping Karyotyping is the process by which cytogene cists take photographs of chromosomes in order to determine the chro‐
mosome complement of an individual, including the number of chromosomes and any abnormali es. White blood cells, cells obtained from amnio c fluid, or cells from cancerous tumors are cultured (incubated) in media containing essen al nutrients, at a temperature that resembles the body, allowing the cells to con nue to undergo mitosis. Mitosis is stopped at metaphase using chemicals, and the cells are ‘harvested’ and then placed onto a micro‐
scope slide, spread out, and stained so they can be seen. The most commonly used stain, Giemsa, stains densely packed DNA more darkly than less‐densely packed DNA, crea ng a characteris c banding pa ern for each chromo‐
some. The cells are viewed under a microscope that is specially adapted with a camera to take a picture of the chro‐
mosomes from one or more of the cells. Once the picture is taken and enlarged, the chromosomes are cut out and arranged in pairs according to size, banding pa ern, and loca on of the centromere. There are 22 pairs of chromosomes called autosomes, which should match up exactly. In females the XX sex chromosomes match, while in males, the XY sex chromosomes do not match. Karyotypes all cytogene c technologists, medical gene cists, or gene c counselors to determine whether a diagnosed birth defect is due to a chromosomal abnormality or to predict whether a fetus may be at risk for a gene c disorder. The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml M. Demsky, T. Mercado, ASHG GENA 2008 Cohort MATERIALS: scissors double‐sided tape karyotyping sheets photograph of metaphase spread PROCEDURE: 1) Obtain a metaphase spread and par ally completed karyotype. 2) Cut out each chromosome remaining in the spread individually. 3) Match the chromosomes with their homologous companion on the karyotype. For example, chromosome #1 is the largest. Its corresponding match should be of the same size, with the same banding pa ern, and have the same centromere loca on. 4) Using double‐sided tape, affix the chromosome pairs to the karyotype sheet. 5) Determine the karyotype and write it in the space provided. The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Appendix II Classifica on of Chromosomes for Karyotyping Chromosomes are arranged into seven groups based on size, centromere loca on, and banding pa ern. Centromeres can be found:  In the middle of a chromosome (metacentric)  Closer to one end than the other (submetacentric)  Very close to one end (acrocentric) Group A: chromosomes 1‐3 are largest with metacentric centromeres Group B: chromosomes 4‐5 are large with submetacentric centromeres Group C: chromosomes 6‐12 are medium‐sized with submetacentric centromeres Group D: chromosomes 13‐15 are medium‐sized with acrocentric centromeres Group E: chromosomes 16‐18 are short with submetacentric centromeres Group F: chromosomes 19‐20 are short with metacentric centromeres Group G: chromosomes 21‐22 are very short with acrocentric centromeres Chromosome X is similar to group C, Chromosome Y is similar to group G The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml HALF KARYOTYPE - DAY 1
Appendix III
HALF METAPHASE - DAY 1
Appendix IV
MRS RAMIREZ
Appendix V
MR. RAMIREZ
MRS. WOLF
MR WOLF
MRS. SINGH
MR SINGH
MRS. JACKSON
MR JACKSON
MRS.GREENE
MR GREENE
MRS DOHERTY
MR. DOHERTY
DOHERTY FETUS
46, XX (normal)
Appendix VI
47, XX +21 (trisomy 21, Down syndrome)
GREENE FETUS
JACKSON FETUS
46, XY (normal)
SINGH FETUS
45, XY, t(13:14) (Robertsonian translocation of 13 and 14)
RAMIERZ FETUS
46, XX, t(14:21) (Robertsonian translocation of 14 and 21, Down syndrome resulting from extra copy of
21)
WOLF CHILD
45, XO (monosomy X)
DOHERTY FETUS METAPHASE
Appendix VII
GREENE FETUS
JACKSON FETUS
SINGH FETUS
RAMIREZ FETUS
WOLF CHILD
M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Appendix VIII 1) 2) 3) 4) 5) Case Review Prepara on Worksheet Did the pedigree analysis reveal any pa erns of miscarriage or inherited disease within your pa ent’s family? Why was chromosome analysis requested on your pa ent? What ssue was used to obtain the karyotype? What is the chromosomal complement of your pa ent? What is your pa ent’s sex? How do you know? Did the karyotype reveal any chromosomal abnormali es? Explain using your pa ent’s karyotype. a. If so, how did the abnormality occur? Are they present in the parental karyotypes? b. If so, is there a phenotype associated with this abnormality? c. If not, describe why not (what occurred properly?). 6) If your pa ent’s karyotype did not reveal any chromosomal abnormali es, are there other inherited diseases in their family pedigree that the pa ent should be monitored for? The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Appendix IX The Doherty Fetus What is the gender of the pa ent? Normal or abnormal chromosomes? Reason for (ab)normality? Normal or abnormal phenotype? Expected to be healthy or unhealthy? The Greene Newborn What is the gender of the pa ent? Normal or abnormal chromosomes? Reason for (ab)normality? Normal or abnormal phenotype? Expected to be healthy or unhealthy? The Jackson Fetus What is the gender of the pa ent? Normal or abnormal chromosomes? Reason for (ab)normality? Normal or abnormal phenotype? Expected to be healthy or unhealthy? Case Review Analysis Worksheet The Ramirez Fetus What is the gender of the pa ent? Normal or abnormal chromosomes? Reason for (ab)normality? Normal or abnormal phenotype? Expected to be healthy or unhealthy? The Singh Fetus What is the gender of the pa ent? Normal or abnormal chromosomes? Reason for (ab)normality? Normal or abnormal phenotype? Expected to be healthy or unhealthy? The Wolf Fetus What is the gender of the pa ent? Normal or abnormal chromosomes? Reason for (ab)normality? Normal or abnormal phenotype? Expected to be healthy or unhealthy? The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Appendix X You are the Gene c Counselor that met with the family whose chromosomes you worked on as a Cytogene c Technol‐
ogist. Your final assignment is to write a le er to the family and explain their results to them in words that they can understand (not in gene c terminology!). Be sure to cover the following pieces of informa on in your le er. Dear Mr. & Mrs. __________________________,  Describe the purpose of the le er. 
Review why chromosome analysis was recommended for their fetus/newborn. 
Explain how the analysis was performed, from obtaining the chromosomes to producing a finished karyotype. 
Describe how the available parental chromosomes were helpful or not helpful for the diagnosis of their fetus/
newborn. 
What did their fetal/newborn karyotype reveal? Were the chromosomes numerically or structurally normal or ab‐
normal? 
If the chromosomes were abnormal, describe in words that the pa ent can understand how the abnormality oc‐
curred. 
What are the implica ons of these chromosome results for their fetus/newborn? Is their fetus/newborn expected to have any medical problems? If so, describe them. 
Were there any other problems detected a er the chromosome analysis was performed? Explain. 
Do you recommend any addi onal tes ng be performed on their fetus/newborn? (addi onal ultrasounds to fur‐
ther study organ structure, etc.) 
What is the likelihood that this problem (if evident) could happen again in a future pregnancy? What is the recur‐
rence risk? Explain why they should/should not be concerned. 
Close your le er professionally and sign your name. The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml M. Demsky, T. Mercado, ASHG GENA 2008 Cohort Appendix XI CATEGORY Saluta on & Closing Sentences & Paragraphs Grammar & Spelling Ideas Content Accu‐
racy Length Capitaliza on & Punctua on Format 4 3 2 1 Saluta on and closing have no errors in capitali‐
za on and punctu‐
a on. Sentences and paragraphs are complete, well‐
constructed, and of varied struc‐
ture. Saluta on and closing have 1‐2 errors in capitali‐
za on and punctu‐
a on. All sentences are complete and well
‐constructed (no fragments, no run‐
ons). Paragraphs are generally well‐
structured. Writer makes 1‐2 errors in grammar and/or spelling. Saluta on and closing have 3 or more errors in capitaliza on and punctua on. Most sentences are complete and well‐constructed. Paragraph struc‐
ture needs some work. Saluta on and/or closing are miss‐
ing. Writer makes 3‐4 errors in grammar and/or spelling. Writer makes more than 4 er‐
rors in grammar and/or spelling. Writer makes no errors in grammar or spelling. Ideas were ex‐
pressed in a clear and organized fashion. It was easy to figure out what the le er was about. Ideas were ex‐
pressed in a pre y clear manner, but the organiza on could have been be er. Ideas were some‐
what organized, but were not very clear. It took more than one reading to figure out what the le er was about. The le er contains The le er contains The le er contains at least 9 accurate 7‐8 accurate facts 4‐5 accurate facts facts about the about the topic. about the topic. topic. The le er contains The le er contains The le er contains 6 or more para‐
5 paragraphs. 4 paragraphs. graphs. Writer makes no Writer makes 1‐2 Writer makes 3‐4 errors in capitali‐
errors in capitali‐
errors in capitali‐
za on and punctu‐ za on and punctu‐ za on and punctu‐
a on. a on. a on. Complies with all the requirements for an informa ve le er. Complies with 95% of the re‐
quirements for an informa ve le er. Complies with 80% of the re‐
quirements for an informa ve le er. Many sentence fragments or run‐
on sentences OR‐
paragraph struc‐
ture needs lots of work. The le er seemed to be a collec on of unrelated sen‐
tences. It was very difficult to figure out what the le er was about. The le er con‐
tains less than 4 accurate facts about the topic. The le er is less than 4 para‐
graphs. Writer makes more than 4 er‐
rors in capitaliza‐
on and punctua‐
on. Complies with less than 75% of the requirements for an informa ve le er. The Gene cist‐Educator Network of Alliances Project ● NSF EHR#0634296 ● www.ashg.org/educa on/gena.shtml