Download Allellic Frequency-Sickle Cell Anemia

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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!
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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