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i t'' .a.r} i'i:'ir):i::r"'::: i. r r'ir.:,:tr -lr:.:,:j. .- Sickle Cell Anemia and Genetics: Background lnformation BaclcgroLrrrd ipfomration to accompany the labs: Allgle-ft-cclttelt:ie-,, Siclrle Cell Attenicr; Dicrgttosis Llsing Re;trictiqrt ilttctll'sis ofD-Ntl c1t14 ,SiclcLe ('e ll rlnuniu !.,als and Genetics of Sickle Cell Anemia Sickle cellanemia was the first genetic disease to be characterized at tire urolecular level. The mutatiou respousible for sickle cellanemia is small!jLrst ONE uucleotide of DNA oLrt of the three billion in eaclt hlpiarr cell. Yet it is elor-rgh to change the chemical properties of iremoglobin, the iron and protein courplex that carries oxygen within red blood celis. million liemoglobin uoiecrtles in each red blood cell (RBC). The proteiri po;tiou of hemoglobin consists of four globin subutrits: two alpira (cr) and two beta (B). These two types oIsLrbLrrrits are encoded by the u eurd p globin genes, respectively. While the binding of oxygen actllaliy occLlrs at the iroti sites, ali loLrr globin chairrs urust work together irr order forthe process to function TSere are approxin'rately 280 rve ll. ^;cl<le cell eirrenria, also l<nown as sichle cell disease, is caltsed by a point mutatiou in the B globin gene. ,l lesLrlt ol'tliis urLrtation, valiue (a non-polar amino acid) is inserled into the B globin chain iustead of !Lrtarlic acicl (an electrically charged amino acid). The n-rr-rtation caLtses the RBCs to becoure stiff and sopretipies sickle-shaped when they release their load of oxygen. The sicl<le cell mLrtatiou produces a "sticliy" patclr on the surlace of the B chains when they are not compiexed with oxygeu. Because otlter rlolecules o1'sicl<le cell henroglobin also develop the sticky patch, they adliere to each otlter atrd ,_ polyurerize into long fibers tfuat distort the RBC into a sickle shape. l'he sicl<led ce lls tend to get str-rck in ltarrow blood vessels, blocking tl-re flow of blood. As a resLtlt, tliose witfi the disease sLrt-fbr painfLrl "crises" in their joints aud bones. They may also suf'fer strokes, blindrress, or cianrzrge to the lur-rgs, kidneys, or heart. They mLrst often be hospitalized for blood trausfusions and at'e at risk lor a lifle-threatening coniplicatiorr called acute chest syudrome. AlthoLrgh many sufferersof sickle cell disease die belore the age of 20, modem medicai treatuents gan sometimes prolong tliese individLrals! lives irrto their 40s and 50s. Tlrcre erre two B globin alleles irlportant lor the ittheritattce of sicl<le cell anerrtia: A and S. lndividLrals rvith trvo nonlal A alleles (AA) have uonlal henioglobirt, attd tlterefore nomral RBCs. Those with two ntLrtant S alleles (SS) develop sici<le cell arremia. Those r.vho are heterozygous for the sickle cell allele (AS) prodLrce iroth nonral and abnonlal henroglobin. Heterozygolls individLrals are usLtally healtl'ry, but they nray sufler sonte syntptonrs olsickle ceil aneuria utrder conditions of low blood oxygen, sLtch as high eievatiorr. Heterozygolis (AS) individuals are said to be "caniers" of the sickie cell trait. Because both fornrs of hentoglobin are made in heterozygotes, the A and S alieles are codominaut. 2.5 nrillion African-Arlericans (1 in 12) are carriers (AS) of the sicklecel1 trait. People'uvho are Jalriers rlay t-tot even be aware that they are carryitlg the S al1ele! oLrt .:j;-i9 Sickle Cell Anemia and Malaria develops sickle cell anemia' In Africa, abor-rt i in I' the united States, about 1 in 500 African-Americans higher is the frequency of a potentially fatal disease so much 100 i,dividuals develops the disease. why in Africa? and fatal disease, malaria. Malaria is characterized by cirills The answer is reiated to another potentially and death may result. Malaria is caused by a protozoan rever, vomiting, a,d severe headicires. Anemia Anopheles moscluito' Whe, 'ralarial parasite (Plusnrocliti,rri rnutis transmitted to.hr-ula."t -bL]l'.^t defeitive hemoglobin become sickied a,d co'tain tirat ceirs red the bioodstrealri, trre pzrrasites invade 'cii., i,fectio,. trappi,rg t5e parasites i.side t5e', and red,ciug greater risk trre AA ge,otype (nonnarhe.roglobin) l'rave a compared to AS rreterozygotes, people with pool' gene the reJLrlts in removai of A alleies from of dying from malaria. oeltr, of AA^homorylot.r cell anemia and have less cirance of cor-rtracting hidivid'als with the AS genotype do not a.uJtop sickle ,ralaria-i,fected regions' Tlrelg,fo-r-e-, BQTH lheagld rurararia. They are abre to survive a,rd r.prJ.r".in SS l-romozygotes have sickle cell anemia' which S alleles of these peoplg ten;1aiU-trt-llr-q"n-o+rt411gl.,' S alleles are removed from the gene pool' r-rsLraily results i,, .u.iy A""iii fiiifrit;;r; advantage on people who have oue malaria is prevalent, tire S allele confers a survival allele is therefore maintained in the population at a copy of the allele, ,,ia ifr. otirerwise harmful S wiil be examined in trre Altc-r-e- Er:equsrrqic-s ar*l Siclile rerativeiy higr-r freqr.rency. This prre,omeno, freqttency in a population to evolutioti' Cell Anelnia [-ab, which relates the change in alleie In a region where regions of Africa is16%.The siclcle cell ailele is also The freqLre.cy of tle S allele in malaria-infected to life' ln areas where malaria is or used to be a ruajor threat r,viclespread irr the Mediterranean and other States, wrrere malariar'rasbeen virtLrally corrtrast. the S apeje rr"q,-,.n.y is only 4ohit-rtheUnited States, but effective mosqLrito control caLtsed the eli.rirrated. Malaria was once comrnon in the United there rras been an iucrease i, the,umber of nralirrial cases ruuurber o[-cases to orop. Recertly, however, resistance to medicatiotr. In Southern californiatherewas because of,i,creased travet, inr.rigration, arrd by local ,rosqLritos! a 19g6 outbreal< oin"orty 30 casei o[,ralaria trarrs'ritted Sickle Cell Anemia and Current Research adLrlt; T'e oxyge' requireme,ts of a fetus dilfer lrom those of an and so perhafs not surprisingly, l-remogrobi, contains two.gam*a !l) sto9ill pre,atar brood cor.itains a special heu-roglobi.. Fetal switch otl' and globin genes encodingy the birtii, polypeptide cSains instead of two aaLriiB cl.iairrs. After trris ge,etic switch works cor-rld allow t.e orres eucodi,g B globin switcrr on. understa*di.g l-row of genes in general and sickle cell anemia in particular' researchers to understand much abor.rt the control variation of sickle cell anemia, sotnetimes with r.ro Indian and Sar-rdi Arabian people have a r-nilder of each perso,Ds rremoglobi^ is the fetal kind' sy,rptours. I' this popuratio, twenty-five perce.t called "hereditary persisterrce of fetai Si,rilarly, tlie blood of adults with an inherited condition th.ese.individr'rals are healthy. Sottre people witli this henroglobiu,, also contains fetal hemogiot in uno i11 effects. Biochenlicai experiurents have conditio. completely lack adult t-,.*o!iouin 1,,d.sti11.show no polymerization of sickle cell henroglobin' de*ro,strated that, i, a test tL,be, fetaiTremoglobin inhibits hemoglobin levels may be an effective treattrent for These observatior-rs suggest that increasing fltal research related to activation of fetal hemoglobin as a sicl<le cell a,er-,ia. Ti-rere are a number of"lines of therapy for sickle cell aneuria: diabetes during pregl-iancy have r-rnLrsitally Sonre inlants r.vhose mothers sLrflered front their blood plasrla. BLrtyrate is a ttatlrral 5igh co.ce,tratio's of tire biochenricll bLrtyrate in I ."ii. fri, ii' ?i;ri,r:I latty acicl that stir-pglates RBCs to differentiate fror-n their precursors (reticulocytes). globin ger-re from BLrtyrate also prevepts the y globin gene from switching off and the B swiicliing oir i1 these infanis, who are healthy despite lacking adult iremoglobin. when butyrate is givel to patients with sickle cell anemia, the y giobin rnRNA leveis in reticulocyt., in.r"up significantly. Perhaps butyrate or_other chemicals that stimr"rlate fetal hemoglotin productiol cor-rld be r-rsed to treat sickle cell anernia. n In 19g3, a drug called hydroxyurea (HU) was first used on sickle cell patierrts to try to coLrld activate their fetal globin genes. By lgg5, clinical trials had demonstrated that HU sickling. increase fetal hernoglobin levels in patientsn RBCs and prevent tl-re cells frorn patients treated witti gU experienced less frequent and severe painhrl crises. However, hydroxy,rea can be quite toxic when used corrtinuously to maintain elevated levels of fetal hlmogiobin and can increase tire risk of leukemia' n Irr 1 ggl, itwas found that alternating l'rydroxyllrea with erythropoiten and providing joint and dietary iroir raised the percentage of RBCs with fetal hemoglobin and relieved the anetnic helps bone pai. of sickie ceii disease. Erythropoiten is madein the kidneys and patierits reple,ish their RBCs. It can be manufactured for tirerapeutic use with recombinant DNA technology. p Mice that have been genetically engineered to contain a defective human globin gene have sy'-rptoprs typical oisickle cell anemia, mal<ingtheur an ideal model for laboratory experinreitation. In 2000, these mice were mated to another transgeuic moLtse line expr-essilg Supran fetal herloglobin. Wlien compared to tl-reir sickle cell parents, the c,fispringlad greatly reclLrcecl numbers of abnormal and sickled RBCs, itrcreased numbers oiRBCs ou.rill (reiLrced ape6ia), and longer lifespans. Tl-rese experin.ients established that orrly 9- l6n/u of hemoglobin ueed be the fetal type in order to ameliorate the sicl<le ceii sy,rptorls, and are ai iprportarrt first step irr a gene therapy solution to sickle cell disease. ! Disclaimer: urateriai was As with ,1apy "hopre-grown" resolrrces teachers use in their classrooms? this background and then severalpeople adapted by and rewritten, culled frorr-i a variety o"f ,o1r.., and has been written, soure of tlie passed orr to tl-re next user. The exact, original solrrce material is not at all clear, but plagiarized. Lrnwittingly been has refere,ces below were used. We apologize if a soLrrce References: Karlsso,, S. Tttefirst steps on the gene therupv pathway to anti-sickling stLccess. Natttre Medicine 6, 139-140,2000. B" Otr' Blouin, M.-.1., Beaucher-niu, H., Wright, A., De Paepe, M., Sotette, M., Blealt, A.-M., Nal<at]]otO, usittg Insights diseuse: cell of sickle C.-N., Starrratoyalpopoulos, G., and Trudel, M. Genetic correctiott I t'rttrsgatt ic tttortse ntoclels. Natr-rre Medicine 6, 117 -182' Keeton, W., arrcl J. CoLrld. Biological Sciertce, W.W. Norton and Co', NY, NY, 1986' r,eary, W. Sickte Celt Trial Cattecl StLccess, I'{ulted Eurly, NY Times, Jani-rary 31, 1995. pp. B5, B8. R. Httntcut Gettetics; Cottcepts ctncl Applicrttiorts, 3rd ecl., pp. 334-335, WCB/McGrar'v-Hill, Boston, 1999. -L.*is, AIIele Frequencies and Sickle Cell Anemia Lab Student lnstructions objective: To observe how selective forces can change allele frequencies in a population and cause I --- --- occlir. evolr"Ltion to Background: Read tlre background infonnation provided in the handout, C en e t i c;-B u ek$ a!t1 cl W1lJotrjAll, Sic.lcle Cel! Anentirt arrcl Introduction: Allele frequency refers to how often an allele occurs in apopulation. Allele frequencies can change-in a population over time, depending on the I selective forces ! shaping tirat popgiatiop. Predation, food availability, and diseaseire ail examples of selective forces. Evolution occurs when allele frequencies change in a population! In this activity, red and white beans are used to represent fwo alleles of globin. The RED beans B represent gametes carrying the globin A allele, and the WHITE beans represent gametes carrying the B globin S allele' The Gene Pool exists in a region of Africa that is infested with malaria. you are simr-rlating the effects of a high frequency ofmalaria on the allele frequencies of a popllation. iaterials: )S reO beans, 25 Hypoth es white beans, 5 containers (e.g. paper cups) is/Prediction : What do you think happen to the frequencies of the A and S alleles as a resuit of the presence of -will rnalaria? (Will the frequency of A increase or decrease? What about S?) Fonn,tate a hypothesis and corresponding prediction. Be sure to explain your reasoning. Procedure: 1' Together with your lab partner, obtain five containers and labei them as foiiows: 1) AA 2) AS 3) SS 4) Non-surviving alleles 5) Gene pool 2' Place the 75 red and 25 white beans in the Gene Pooi container and mix the beans up. 3' Sirnulate fertilization by PICKING OUT two nallelesD (beans) MTHOUT LOOzuNG. Ior every lwo beans that are chosen from tire gene pool, another person will FLIp A !COIN to detennine whether tirat individual is iniected with malaria. 5' Using the table below, the coin flipper tells the bean picker in which containers to put the B beans. tr$t ififected fslal*r!* {l-{*ade} Fl"renotyp* fien*t5iBe t I dli:}, AA (Red-Red). AS (Red/White). No sickle cell disease. Malaria susceptibility. Die: place in Nonsurviving Live: place in AA No sickle ceil disease. Malaria Live: place in AS Live: place in AS Die: place in Nonsurviving Live for a brief time: place in SS resistance. SS (White/White) Sickle ceil disease. 6. Repeat steps 3n5 until all the beans in the Gene Pool 7. Record tire results in the F1 are used up. ctIP TALLY table on the data sheet. At the eld of the round, COTINT the number of individual red beans (A alleles) and white beans (S alleles) in the containers labeled AA and AS. These individuals survive to reproduce. nlCOnn thor" numbers in the Fl TOTAL SURVIVING ALLELES table. Put them in the gene pool afterwards. 8. 9. Because SS individgals do not survive to reproduce, move all beans from the SS alleles container into the Non-surviving alleles container. STOP AFTER ONE GENERATION CHECK WITH YOTIR TEACHER BEFORE GOING ON! 10. Repeat the procedure for the F2 generation. Record your resuits in the F2 CIIP table and F2 TOTAL SURVIVING ALLELES table' Data Sheet for Allele Frequencies and Sickle Cell Anemia Lab (All students t'reed Fl CUP TALLY: to record the data in their notebooks.) Put a mark for each bean next to the appropriate cup' TALLY :ron-survlvlng Fl TOTAL SIIRVMNG ALLELES: Nr-rmber (very rmportant to record) of A (RED) alleles surviving (Count out of AA and AS containers) Nr-rmber Pr.rt tire of S (WHITE) allele surviving (Cor-int out of AS container) sltrvivors in the gene pool and create the next generation. F2 CUP TALLY: Put a mark for each bean next to the appropriate cup cup' Tally AA AS tr: t'lon-srirviving F2 TOTAL Nr-rmber SURVMNG ALLELES: (very inrportant to record) of A (RED) alleles suruiving (Count olrt of AA and AS containers) Nr-rmber of S (WHITE) allele survivilg (Cor,rnt out of AS container) Class Results 01 the ciass overhead, record your number of A alleles surviving for the next generation and number of S alleles surviving thc C1ASS frOM bOtlr thc F1 TOTAL SURW\rING ALLELES ANd F2 TOTAL SURVI\rI}IG ALLELES tAblES' ThCN TCCOTd totals below and calculate the frequencies using the formula below' Using the fornulas beIow, calculate the % a1lele frequency for each aliele in each generation: TotalA x 100 : % Al1ele A Total S x 100 =%Allele S Total A+S Total A+S ss Results Table Parents F1 F2 NS Total Allele Frequency Analysis Questions Allele Frequencies and Sickle Cell Anemia Lab Answer in compiete thoughtsl 1' what do the red and white beans represent in this simulation? What does the coin represent? (See background information.) 2' what do you think "allele frequency" means? How are allele frequencies related to evolution? (See background information.) 3' What are the "selective forces" in this simulation (flre forces changing the allele frequencies)? 4' what was the general trend you observed for Allele A over the three generations (did it increase or decrease)? What was the general hend for A11ele S over timez was your hypothesis supported? 5' Do you anticipate that the hends in question 4 will continue for many generations? why or why not? 6' Since few people with sickle cell anemia (SS) are likely to survive to have children of their own, why (S) been eliminatedt lHlnt: whai is the benefit of keeping it in rhe popularion?) hasn[t the mutant ailele 7' Why is the frequency of the sickle ce1l allele so much lower in the United States than in Africa? 8' Scientists are working-on a vaccine against malaria. what impact might the vaccine have in the long run ou the fi'equency of the sicfle cell allele ini.tricaz (Would it increase o, JL.."ur"? why?) Overhead Master for Class Totals Allele Frequencies and Sickle Cell Anemia Lab Class Results Parents A S F1 A F2 S A S Allele Frequency Total the column for each allele in each generation and calculate the % allele frequency in each generation: Total A x 100 = % Allele A Total A+S