Disease Gene Identification 1

TABLE q.rl
SomcoftbMortCommonSFngkCmcTtabin~-
Dhemse Effect IncMence of Disease
I
Caused by Rec& An&
Thalassenia (chromosome 16 or 11 )
Sickle-cel disease (chromosome 1 1 )
Cystic fibrosis (chr~rnosome 7)
Tay-Sachs disease (chromosome 15)
Phenylketonuria (PKQ (chromosome 12)
Reduced amounts of hemogbbin;
anemia, hne and splm enlargement
Abnormal hemoglobin; sickle-shaped
red cells, anemia, blocked circulation;
increased mistance to malaria
Defective cell membrane protein;
excessive mucus produdion, digestive
and respiratory failure
Missing enzyme; buildup of fatty
deporit in bmin; buildup des?roys
mental dewtopmmt
Missing enzyme; mental deficiency
1/10 in park of Italy
11625 African Americans
112000 Caucasians
113000 Eastern European Iews
T /'I O,M)O Caucasians 4
I
Albinism [chromosome 11)
Missing enrymg; unpigmented skin,
hair, and e p
1 /lO,MM in Northern Ireland
I
I
I
Catrsed by Dwnimnt A/&
Hypercholesterofemia (chmos~rne 19)
Huntington disease (chrwnosome 4)
=-\
GET
A
1
I
\
% 'k.
- - - Figwe 9.1 S. Camparison of tfie steps in - -
FUNCTIONAL CLQNlNG
functional and positional gene cloning. In
mdicional functional cloning, the mdv of gene
function precedes gene identification. In posi-
(-
tional cloning, Fne mapping comes first, even-
--
/'
mally leading to gene idenutification. Gfter the
WNCtlON TR~
pcne h hen idcnrifid, dus then pmia an
Le.-ttj
A opoonal study of p e function. The solid arrows
indicda required steps in the approach and
dotted a m indimre optional steps. (Adapted
t?om Collins FS. Pmitional clonin~ let's not
call it reverse anpore. PJat Genet 1992;1:3-6.)
POSITIONAL CLONING
Fi gum 10.7. Progress in undmmnding
human disease at the mdedar level hllows
a predictable pat-
from top to kt-
torn in this d iap, dthough the time interval
inwlved may he years or wm
demdes. ?'he advenr of the Human Genome
Project prratly amlerates the abilig to identify
human dirnv penes hy rhe paitional cloning
smregy. After a gene has been identified, preventive
medicine smtegies can h designed for
chose individuals found to be at hgh risk. h the
long mn, however, the greatest prumir of ~ n e
dimvery wilI be the development of new and
more effectjve therapies.
I - --
M*ming protein that remows choleslerol
frwn theblood; heattattack by age50
Pmgressiw mental and ~eurolqical
damage; neurakogic disardm by ages
4670
-. . - . +
- --
DISEASE WITH
GENETIC COMPONENT
I
1 1
MAP
CLONE GENE
A CCELEFIR TED
BY HUMAN
GENOME PROJECT
DIAGNOSTICS
/ BIOLOGICAL DEFECT
J
PREVENTIVE
MEDICINE
---
GENE THERAPY
f 1122 F mh Canadians
1 J25.000 Caucasians
-- -
DRUG THERAPY

,-
1. Black urlns dl-
-
2. Mendallan mcwmb
A KU
. I
j : Cases of hy%Mlzatlon -
. .* m.
I .'
\
9. Proposed enzyme deflelency
Homogentiskc acid
1 EnMHGO --
Maleylacwtmcetic add
8. eONA finds gsns h~ k gmmlc Ilbmry,
4. AKU gene mapped
I- I- Till 1 1 1. I rl-
I- "I.
9 P
C h m
3 ('-'ILz
3s2
AKU
C
I
I
-I l. re .--
Y
HGO gene (1 4 exons, 13 Intrm)
-
5. HGO Idatsd Crom fmgw A~pemIih~ 10 *-Primer 12 --
I
9. PCR a! mena 1.0 and 12 find mutsfit sltm.
- .:.: -
, HGO
-
. '?
--t
m
- -bz~300G ---
- - ---7 m s
4-
HGO gene
-
8. A-Ius HQO finds h um cDblA. 10. Inmncs oi muteHons
Figure 11-22 The steps in unraveling the biochemical, p Mc, and mnlecalar basis of alkaptonuria.

Empress
Victoria
Albert
1
Victoria (1 81 9---79Dl)
Kaiser George Princess Frederick (Alexandra)
Wllhelm lI V Irene Alix Tsarina
Nikolas II
Alice
of dthlone
Vjctoria Leopold Ma1 mrr
Eugenie
(wife of Alfonso XHI)
FIGURE 15-72 A partial pedigree of hemophilia in the British royal family descended from Queen viaoria. The pedigree is
typical of the transmission of X-linked recessive traits. Circles with a dot in them indicate presumed female carriers heteroryga~;
for the trait. The photograph shows Queen Victoria (seated at the table, center) and some of her immediate family.
Iim
Figure 9.4, Psdiffse 3-g
of
the p e fw -0nic-b to the serrttm
laus. 1-d~- with rdrd~h&
arc a f f d with xurmrd dominant myntonic
dysaophy. In A are shown rht mtm
phenmypcs (+ or -) for tach of the inclividu-
PIS IR the pli- Gcnotyp ore infmcd in
R wing the fact that rk Sr allclc b d b t ,
so hat + individuals are either S& or SeJq
and - individuals murr he dsc. Tbc po-
typesofall ofthe + indivictuabuqx form-
- mu bc infcd hy the ncrmity of
Mendelian inhctinm* at the sccretor locrrs.
Thus. for mrnplc. individual 1-1 must k
Sdw, nw %, 1-usc he h three seJw
06qn-h~. I+M of B show rhat the myntonic
dystrophy p e and the Se dele at the
sccrttor locus am appmmdy linked in this
pedim.
- -.

(a) Key = 0 Norma! lemab
O CRMW ( twtemw~) [wrmla
0 Normal male
AHectod male
I
Albert
ck-@
Warla
(1819-1901)
1
Innclivr! IX - Active lX
I
Fnclors
XI1
XI
IX
Vlll
X
v
Prothrombin
Fibrinogen
Wlld-Qpe
person
Hemophllfat
t 4
+ +
+ +
+ - -
+ +
+ +
t +
+ +
Fibrinogen - Fibrin
(d) Purlfled
Faclor Vlll
I
Obtain arnCnb
acid sequence
I
Reverse translation
into coding sequoncs
1
Synthoslxe degenerate
oli~nucloot~dos
I
Probe llbrary
-
I
Flnd Factor Vlll clone
1
Structure of gene
AG lTG GCC TAT CGG, ,,
.,..,..,...... ..,...., ,.,..
1
....--.
TrhuCcQaaTm
TITMCCWATAOCC ...-.
TltiMCp( iGATAOCC
-
as datmilwld fmm 11- I -L- 1-1- 1.- _L--I ...-
CIIXIP m o 20 no FO nn IM I'Z~ 1.10 ~tn ~ B O m - - --
Agum11.16 How~~~drknsdtha~uI.A~.(rr)Aprdlgmdthcrqrslfaml~desctndcdtnwn
Vllccll Wctona. 'This tarrltly trw uses !tic rl,sndard (nrdrqrw rymbols. (b) fhc- bkal-clotthq cbxade. Wwl dmaqe indum a amde 01
m~rymatic emts Itrat ronvrrt inactiw- lac ton lo active l~tcm. 71rc c,rxaclr r~wlls In thc trilnslnrmaliun of hbrinogen to tlhrin and Ihe lonnathm
of a clot. (c) Rid tsls can detcrrnlne whether arr active lonn of each f,ictor cnvokd m the clolt~q cascade is prcsenl. Thc rmult. of such
analws show that many hcmophil~~m, such as thme lwmd in Qwm Victoria'c ped~qrcc, lack an ac!we Factor Wll In Iheir blood. (d) Research
purllied Factor VIII, detrrrnined ~ ts amiwcid sequence, u d thlr ininmation to infer all pxsible dqwmatc coding qomccr. constmad
oliqol~uclcollder tor a rcq~on wllh rn~nimal degeneracy, probed a q m i c library with Ih~5c oltgonuclrnbcks. and obhincd gmrnlc clonr.~ d
thr radar VIU qm. llwy qu&
Mt- clomr to dclrrmtrw rhp qenc's arlual cdiq wquwe.

Fkpm 7.29 Honr mutathms modukta Ilght md eolar
pnrapth. (a) Arnincwcid substllulrons (bkk&Is)
that dlmrupt
rlmdqnin's Ihree-dimenwowl structure result in retinitis
pgmrmtourm. 0th whrtihs diilnnh~ hdopdn's clcmltw
-
to lyht cauw nlght Mlndness.
----
MurATlON
SEARCH
. NORMA!.
M
faENTlFICAlION
I TFIANSCRIIT ffim
ci Kt"
YACa AND
mcs
Figure 9.31. Schern~tic of &c gencrsl appmach to paaltionml cloning Thntlph a
un~r(necywgcntric rhnormnlity ~caqionnlly pruvidcsn ahoncut, the di-se getir isuruallv In#.
ri3IIy mapped hy l~nkapc analysis oiaffectccl I;imilie~ using a laqy hmry ofpcrlporph~c I)S \
marken (a pcru)rnc scan). Snriietimcr n likely candidate gene i5 qitickly idcntitied hased on 11s
function and known Incutinn r~car where rlic ini~ial ditmc Rcne has bccn mapped (lowrram;~).
7 h~r sl~orrcut is ohrn rclemd to ar rhr pori~ional mndldatc appmach.

" --- . - .- '-J-u
--nnq-mtamqmi
W 4 em -1- *pwJu!+al
d~ pur Llolr pw Rnybr palepuwtun
S Pm .5 4 1 m-1 Ypralwokn. pur uwoq
kfi prr lado .u*lrplkr WBq qllm ympHIpn
--
u'PIlolParlv*uluadRO#PWIUSN4odur,mkn
-w 9 .C PW r 'I ~mxa IU~PKI+~I IQSN Mtx,
-09 -6 pr* -6 ~?mtpul wry filit~q
m b m WJW* =)a+
(sp+rur
HH4 k payo)W amqrplq .Fan tuuwd3 &%I.'
ga-q -n mlranlarl mm PIP PW ~undrpaq
sf% fiunmclr dm P~~LKOJ,-JW~~ 'e .ewW
.LgpSWYmRWt ul wcvlmnlu barn L .61 j
**'

-. -A-
Normal
H O m O r y tw ~ 1.5-Mb
duplicetlon-smem3 dl-
*
-
Activating point mutntinn
,
:zz+
,=~
duplicated mglon
Figure 16.k Gene dosass sffsch with the PMPZpens.
- -- -- - -
- - L
MOS! pationrs with charcm-M~rle-Tooth disease are heterozygous for a 1.5-Mb doplicdon at 17pl1.2. including the gens for periphersl
myolin prateln, PMPZZ(colomd box). k pallent ~ O ~ O ~ ~ Qfor Oihe U S dupl~cation had vary severe d~sesse. Some pdents have only two
-- - ---
copies ot tho PMPngene, but one copy cvrrles an acl~vatlnq rnlrtat/on.
n#rdrWOrSchnakRP.*rr*.lhndrn
Lnvl nntrR~ nt wmparath w mk wldm
~GtnnflWAngnlmtnRACMYA~~ravmrb
rcrr~hu~n~rnn~tm~pabrcmmrm.
hnvm bnn nrtnrwpl In tNmm fmn rhor mq~w
rrirlnq the rmmlnnl M ot t h pslm (prm) to u rn
rrprmrnrlm t b trrmlml rrrd of the q nrrn (qtrr).
Th.tmrOlDNAh~.*lhs(lnrm+r*lIhe
taH DNA h LnhrM wRh mY dyr. C ~ml WklfMbn
lhsvrl wlfl prrmrtrnn ydknucokr. Frm orrnmrrrinl
In lhr. ?r* DN4 wHlnwn Inn r d Jqnol. ml
*'I IPwy of wnomlf mmwilrlnl Mll pr- n ~pmn
In tfm CGM +M, Um mard DNB h Imm
b frfmh?. 7M p%wm.autonmnl rwlhm In X-n ma
Ip+m nm wmlca wllh!*m~~rl~mtlmh.

(a) Pceparr: mlcmerrey.
Human chromcwome
(b) Prspate genomlc DNA mmples.
Conlral ONA
test DNA
I hbId\h
lhrorescenl dye
-- I
f-9
......................................
......................................
MTw logelher nnd denelure
......................................
I
......................................
(c) Incubate mlCrm~
...................................... - ,
-----..
_.-Y
- -
Wlth cambtned esmplas.
' I . , .
6 I
-\ /'
Human chromosome
K* pin? db
- i \
~ p (3.35 . Camprnttvt MnJI M(au duplhtkons, del~s,
and neqJoI@. (a) MC d m npmrntinq
tl~t~~tnan
cmtd qmm* sarnpk i~ labd with a mnd cdar dye (y~lbv).{c) The twn mmph n mixed tqlhrr, dmaturwl, a d ihm incubat4 on
~h mrrmmy. (at) A~nanatd annbis of each spot m the rnW.ly &t&s the h~ta nt Vlc two dywf pDb" !hat hyb.dke. Qmqs YUIIcates a
1.1 ratio; athn rdm indl~ba d~Wbri {0.5:1 ralio; p4k1~) a dup6caIicn~ (131 ralio; &) of WC chw u(urtun in th, test sampb.
genrmr arc yatld in o ds on(o a micmanay. (b) Thc qnamir s.nnp!e lo b tend k labelol wlh ar cob dye (me, m, and thc
-

dsfactiva Onne
caunlng d~sse*a\
11
SNP marknr on
thlr copy el
chrorno$oms only
chromosom~ pnlr In
mothar with rllsa~se
I
TCSTS PI
I
-ma chramosom~ pafr
in disoa5a-frao f~lhor
I
worm
: 1 ' I nann causing dlaoaso 1s colnhnrltod with SNP markorlrom dlseawd mother In
76% of tho dlsenaAd progeny. If thle same carrelstlon In ohserved in olher hmllias that have ban
@xarn[ned, ll~n gnnn cnuriln~ dlscase la mopped to lhls chromoaoma close to Ihe SNP. Note tho1 a SNP
(hnt La either fmr sway from tho Ortno on the mma chromorcomo, or lmled on fl dlfTarnnt chromolomo
rl~nn rho pno of ~ntoresl, will br: cn~nhorlted only 50% of ihn time. -
--
-
1
Figure 8-59 Csnetlc linkage anah
using physical markers on the D N ~
to find a human gpne. In &Is PY.~~~,,,,
one s ~dit~ the colnherltlnce of a spec#
IC
human phenotype (here a genetic dlqp1,q
with a SNP marker. I( Indivldualr wr,o
lnharlt the drsexre nearly always inhwr
particular SNP marker, then the gen*
causlng the dlseass and the SNP are llko+
to be close together on the chmrno~%,
as shown here.To prow that an ohw
Ilnkage Is rcatlsllcally 11plflGinc. hundmd,
of Indlvlduals may need to be axamlngd.
Nore that the llnkge will not be abtolu
unless rha SNP marker k located In [he
gene bslf.Thus, occasionally rhe SNP
ba separated from the disease gene by
melotic cmsslng-ova7 durlng the
formatlon of the egg or sperm: thlr ha *
happenad In the casa of the chmplr
on the far rlgh~whtn w~rkly wEth a
sequenced genome, thls pmsdurp wuld
bc repeared with SNPr located on either
sjde of the Inlrhl SNf! until a I m
colnhedtance b found.

Rgwm 1.20 Cystk flMs: A mcedve condltlw. (a) and
(b) ~llurtratr the pvdlqter.r ot famiti- with tyllc fibrordr. In [a) a
cnnssmplneous njat19 In qencration V hrwcen unaffected third
cdm (V-1 and V-7) who an! cmncn (hFt~.rorygoter fm the dominant
nomral ale CT* and thc rccmrivc dka* all~h. CT) prcducm an
aff wlrd Ct CF daughter (Vld). Simihify, VI-1 and Vf-2 are canien and
Ihr~rt CFCFhomcrqqaus dauqhtcr MI-1) hm llw diwaw. I1 Is probablr
tlk~t irrliuzlh 11-2, 11-3, 111-2, Itl-4, N-2, and N4 arc all canim or the
=me CF alMr inhetitd fmm 3 common ancestor in thb Ivmt
gmemlion. W tannot detmiw which of these Ioundrrs (1-1 or 1-2)
was a can&, sa HIP must d p j i ~ their t ~ CpWlyp~ as CF'-, kauw
Ihc CF akbe Is r&lMy ran- In thir population, 11 IF like that mart
unaffeed h&idwls unrclatd by bld
(11-1,114,111-1,111-3, IV-I,
and TV-3) am CT ' CF' hom?yqotrn. Ihc q~notyp~ of the vrnaining
unatfedd people (Vl-3, Vl-5, md VII-2) is uncertain (CF '-1. (b) Two
familm in which car& parenu pmduce multiple childwn with cystic
fibros~x dernrmrmtc hon;ront;ll pallcmr of inhrrilame. W~thoul furth~r
inflmnation, thp unaffPctPd chDdm in each pdiqr~e must be
rrq.mkrlashminqaCF'-vtp.
n w
A HOW molawlsr rnatWns helped locale the wane far cWlC
flbrnl5 (CV.

-? U u
I 80 kb :a; 5
-
c' " m,
-, -8
r. e-*-,-, EukRry6llc DNA ,. - 5 > L .
-.
I/ Cul with EmRI; lnserl belwccn ).arms
-------
b
Lu-d
""'"" /
Scmn with
u
gone probe A
L -=-.-
--
:.,.,I- . .. ;
J.)
Subclone small fmgnent
' : Ramen Ifbrary
i/
,.- L!i 7 , .':
Adlacen1 clone 2
I!
Subclone amall fragment. ete.
ln 4 . "
-r -..- - I;?
a .2 n?
----m I -I-., a- r~n' K!.
- -"-*-.-AL. - -.- .- 2 2.: + 1
. - -2G ,-h-A* ;r'z
- #
DNA Fragment
-7, J
- 1
I
Slartlng polnt
0
Cut
Cut
r /
1 1
1
.-.- - . .-
Bldreeflonal walks
L- ----- A- .-. I ?
Use as scmd Jump
starling point.
figure 74-79
Manipulsfimg cloned ~ennrnie frapmcnlx for
chromrwnmc jtrmpin~. a mbdiftcd lype or chmmrnnmc wnlkinp that
can hyplrs rtpionu difficllll tci clone. sctch nn those conlaininp.
lc~lilivc DNA (see tcxt).
-

Clm Into EcoRl cut plasrnld
Trannlorm bacfnris
Select for fi

(a)
Chmosome 7 267cM
' barid
I
" t
4 candidate genes I CFTR
(C) (4 - Clone
showlnn
m hybrldlzslion to CFgcno ,
kb0 SO 100 150 200 250 300 350 400 450 5Jh3
". , -
-,.I-
.-'7- .-. . s CFgene "
.c
Le)
Exons
~i 14b
1 23 4 S C Q 7 8 0 401112 13 \ / 15 161Tal7btl lBZa21ZL~ 24.. I
.................-.-. ... . . ........ -
.I.............
m.7
. . a,....
.. ..
sm..
.I (I0 ..D
I..
....- ............... ,
.. ". ".
111 11 1 1 Ill111 1 1 1 Ill II IIII!.-;
Mutations
F
285- Corresponding wilcllype ~olded pro!n:n
polypepHdo structure
Insefled Into
membrane
m Y.
75 4.zL
Flgura 10.19 Posftlonnl clanlng of the cystlc flbmslr gene: A Mew. (a) Linkage analyrir places the Cf locus on chromosome 7. Th
idlogram of chmrnoson~r 7 show< lhe positions of markers-met, XV-2c, KM.19, and J3.11-that del~ne the I- to 2-eM region within which 1
IOCUS must lit. (b) Wcllklng from a liriked marker to the CFgene. Us~ng the closest linked marker (XV-2c) lo pmbe a cosmld llbraryof tkgMOl
(with avcrage insert sizes of 30 kb), re~earchcrq putled out clones that they characterized. Thy then used the ends d these clones to probe Ih
library again. Thcy rcpcatd thb pnxcls many tim~r to obtain n contig of 2RO kb. Withln thir 280-kb region, heyi id en tilled four tnnscription
units. These marchers punued thc cloning of thp Cf gene before the development of the YAC vecton that make it pos.rible to prducc law
ln~crt genomlc libraricc. Tday It would be easy lo obtain this entire contig on a single YAC clone. (c) Northern blot analysis reveal5 that only c
the andidatc genes is expressed In Ihe Iungr and pancreas, both affected tissues In CF individuals. Thls candidate (now known as CiTR) is not
~xp~~~scd in thrn brain, liv~r, or testes, all Ussues that are not aHccted in discad individua!~. (d) The 5'end of the CFTR gene was located a[ 01
end of the original 280-kh contig and found to extend ovsr 350 kb of DNA. (e) €wry Cf patienl has a mutated allrle of the CFTR gene on
her or his chmmosom~ 7 homolqs. Tht. locations and types of mutations uncawrect in d,ifrr~nt patirnrs are shown und~r the diagram of the
qcnc. The normal gene encod~r the wild-type protein.

Normal
[DNA ..,OM AAT ATC ATC TIT M T OPI TCC...I
Protein Glu Asn lle Ilo Phr Gty Val 9f
Posl!ion 504 505 508 507 509 509 510 511
-9.17 Dlrwt-dUnmnost-qek
fflrrwl~mhThr C F ~ ~ ~ t ~ a ~ ~ 2 5 0 , 0 0 0 h m e ~
and Is qan17Fd In 27 Pxons. It encode\ a prolein wilh 14AO amino
a~irJr. (a) Thp nlost c m m dtwase-camlrq mulalion in Che C% cjm
nackhkmolIhmehine~on 10.ItaporsibktoamplilythE
rt-qlon containinq the ril~
of the most common mutation by K R d
r'lvldr the PCR prducls into twu alquols. You thm blot the al~qudlr
expressing 7 7
onlo fiftpr papm and pub with AOI lm th~ mld-type and muwnt
RNA paharase
nll~kr, Thr AS0 f a thr mrtant all& Cmtm by the of I t m
barn fmm tho AS0 f r thc wild-lyp allele.
T7 RNA pab:m.e nlrno
I
PrCURE 12-11
Expression of the cyjtic fihrnzir gene (CF) in a vaccinia
vin~s vector. To provc thnt n wild-tvpc CFgnc could
correct the ion-rranspon rlcfrct in cells froni a cysdc fibrosis
psricnt, rhc wild-rypc gene urns expressed in rhcsc cells
using a rwo-campancn~ vaccinia virus cxprcgion system.
Firsr, the rclls wcrc infectctl with n specially cnginccmd
vsccinia vinls rhsr carries rhc gcnc cncocling bncrcriophagc
T7 RNA polymcrnsc. l'hc infibctcd cells I~cgan to producc
the RNA plymcrasc. Ncxc, the cclls wcrc rrnnsfcctcrl with
a plnsmid carry in^ the wild-vpc CI: gcnc dnwnstrcam of s
becteriophagc 'l'7 promoter. The 1'7 RNh polgmcrasc
cllicionrly transcrihecl the CF gcne into rnRNA, nntL the
mRNA nns rr~nsli\tcd into protein. 'l'lle resulrinp: cells were
nnalyscd for rhcir ion-rmnspon acrivity and found lo hivc
normal activity, proving that the clnncd CF ~crlc could
correct thc dcfccr in rhc parienr's cells.
Imndwt D M
info cells using
lipofw6ction
Harvest cells ,-
\ CF mRNA
CF protein
Anolp srpression
of CFpdein by gel
clecfrophorcris
Assay
imchmnel
activcty

Original markers - M3
lo oblarn rough
postton
I
Exten! of map position ol
diwnse locus fwnd lo
linked lo M 1 nnd M2
with each marker
(b) H-m-H31-l-421-K1 / ltvacdosast
mnrkors thnl
NW markers used hf higher- hllneate di5-w
resolulion linkaqe analys~s locus
M4
Campare andidale
from NO ~ ~ U J01 I S people
--+- Normal indivkiuals
with phttn(rm
lndivWual9 with
mntanl phwtype
Rgum11.17 Pt~ftkmdclonlnq:Fran-to
chmmosamal Iaatlum to gulw gam. (A) C)maqmrn d a human
chrc~rnornmc wrlh four rna~kcrh -MI, M2, M7, emf MLud
In !he
11nk.iqe analytls of a dtwarc phcnoiypc. Each markcr prwid~r 7inkaqe
covrraqr" of a portion of thr chmlr~osome. Tlic ~ ~ of COVCT~JP q b lor
each marker IS thp wgmn wlthln which lrnkagc to a disease k l r cwM
be uncwcred. In thc t~ypblbltcal study ~!Imlml~d hew, Itnkaqc. has
hen drtcctd lo adjacent markers Ml and M2. This ruqqesu that ItK
gcnc rmponr~ble lor Ihp disease 111-5 between Ihow mark[-rs. (b) Will)
Lhts ~nfatmnlion, an Iwt~gator rwld typ addiiisnal mnrkem that IIC
between MI and M7 to psrlron Ihc diwav locus wll\ Iliqh~r
rrzolulicm. (c) Looking for candldatc pm. Anabir of Iljc nqim
betwffn rccomhirwt~on siter !hat drfine the smallrxl drca wlthrn
whrrh tlic drwitse Iocus can lit- rtrwld rcvral the prPscnce ol randklat~
gPne.r. (d) Flndrng Ihc c m t cand~dalc through comparisons of Lhp
Tlnlcturc and cxpr~sslon of rach canrlldate qenr in many dircarrd and
nondiscased indlvrduals. A corrclalmn between a mutant rlructun or
exprerrion lor J piirtlcular ciindlhtc gene and the diwau ph~nntype
can pmdc evidPnca that a lpnrtrcular gene is mpnnsiblr fnr ttw
dlreaw phenolypc..
-
I 7
TABLE 1 1.2 Complexities That Alter Traditlonnl Nkndensn Ratles
Prohlern In Obre~ed Relatlonzhlp
Category of Complexity Between Genotype and Phenotype Changes Necessitated In Mapplng Strategy
r,
Incomptrltc penetr,incc
Rhenocopy
Gmrtic hetemqmeily
Polyrl~wic determination
Di5Pl~r genotypc can wrilr in dlr
individual who does not expm the
daease phmotype
Disease phenotype can b expressd by
an indgdual who dws not hrwe the
disease gcnotypr
In diff~wnt famllim, diffmnt disease
genotypeq are mponuble for the wme
diwase phcnatype
Mutant allelm at mow than onr locus
influence exprenion of Ihe disease
phenotype n a singk ind~vidual
Ellminatc nondircared individuals from analpis
-
Proqram computer to flag unexpected carps of
ruppowd double r~comhinatlon wen& that flank
a pitalive diwarp locus
-
Divide complete set of dis~ase-tranrmittinq
famllies into subgroups ( h s 4 on various
parameterr such as nveraqe age of onset) and
perlorm linkaqr analysis separ.rtrly on each
subgroup
-
Prqnm cornpuler to search for complrx
palt~rns of arsmladon ktwepn the d~rmw trail
and mulllplr loci
J

J7J (73 DeceflsM
13rcasl cancer
@ ovarian camr and doceasad
Q) Other cancer and doceasnd
Ftgum 11.23 Cem?lk httemgcntlty: 1IYMstbru M dWcrmt Id
cam gtvt CCK to t.k same disease phenotype. Roth pedi- in
lh~t liq~m rhw wldpnce of !he tr.ln%mrssron of a domlnant mutatlon mth incm~plcte pmctrance that raum t>rp4r\l rdricrlr. LinL1,rrpl analylir
shtnvc Illat Ihr mutil!iOn In the lint ped~qrw mrdrr on chmmorornc 17, whcrrar thr mutation in t k second p~d~qrcc it en chronio~urnr~ 13. The
fint family ir qmling a mulanl BRCAI all&, wh~l~ the %wand farnib is sqwqatlnq a mutant RRCAZ n1k.k..
Lewnd
Male 0 Female
a ZI~kcd
0 Brensl cancer
I:) Ovarian canmr
(1 B~lolnnl broost cancer
~gu&0,14 Llnkqe mrpplng of the b t uncar gene
BRC.41. (a) Podan ot a !my prdqrce showirq Iritnrmrrion ol the
hw,i\t cancer phtyp~. (h) Obla~ninq incr~a5inqty qreatrr rpxnlut;on
in mappilrq Ihp RRCAI ku5. Rwarckn lint rnapp~rl l BRCAI ~ lwur
In 11nk.q~ wilh smal markers (Dl 75750 etc.) .v~ild w~rt along 1b
lotq drrn of lu~nwri rhronwmm 17. Following dwnonstnl~rm d
IinLcy to Ihit rqon, th~y ranid out hqhcr rmlution mapping 4th
(b) Chmrnasome 17
3 markro and gpnrs looted btwcren Ihc two oriqlnal n~arkcn
m
Marker Cnndiats
74 t 1
Dl 75250 and 01 75579, which defi~w Ihe extent of the reqion within
Flcmffj
which BRCA l had to lii.
21 3!
PI. 1
31 1
13 ' ' !‘-:
-

FAI,RII
Candidate genes
IEII DC:~ i DCI e ~ci.1~1 AIPCICH.
- ---- - -
OlfS5as P
r 1 1
PW DWT,7'il UTl04UTV3 17-HIiO Dl 7W4R U fa W U13BS MS
6 I I 1 I Ir-'I 7-7-
W3ZU DnmM K ? m WRA I*rRA I fw
Dl7SST9 CPn1 DI7SlA1 PPY UT61 RNG? D17SB55 CI~lR' DITS?76 A m UTE? Dl-6 lJWll7S ZSkm.lkr Dl?%11~7 I)lW CSf3 U1;10'.
1- t- 1- L~rnlts of location lor BRCA 1 4 --I 4 --I
1)
kpm 16.15 Physical MuqqAng of the BRCII Loas. Wlh many family pdigrccs and mark- in Ihe RRCjll reqbn, mearchen locallzed
thi7 BRCAI 1mus klwcm Ilkrbly rmomhindllon vies that delineatd a mion lcs~ than 1 Mb In Imglh. Aftrr cloning ih entire mian into a w w
01 owrlapping YAC dnd h~lt*~i~)pI~ilg~
CIOIIPI (41 YACS and 11 phaqes), they idmtiiwd ~IW candidar~ gmes tht seemed to be cotransmitt~d with
Ihr RRCA 1 mutat ion.
- - - -
+ All complex mltr
$
/
d
i
90
80
70
ria
flg. 1, ldcntlflcatlon of genes underlying human Mendellan traits and genetically complex tralts ir
humans and other species. Cumulative data for human Mendelian trait genes (to 2001) include el
major genes causing a Mendelian disorder in which causal variants have been id~ntlflcd (58. 59)
This reflects mutst~ons in a total of 1336 genes. Complex trait genes were identllipd by tht
wholegenome screen approach and denot~ cumulative year-on-year data descrihd In this rpvlt-w

-
New mulation (M) In (b) Three reglons (c) Collect DNA samples
encestral population
ol haplotypes from rare lndivldunlr: wHh
dlsease and a control group
* I B l C conlainlng an equal nurnbr
without d1Seas0
1
Disease group
-
I Compare fmquency of each ]
I&
haplotype lrom each ragion
40 genecarlons batman tho disease group
the control group: The
only slgniflcant dlftcronco Is
M !
e....,... I .- In the blue haplatym ol I
uulllrul MIUUC,
I I and
region 0. which oihiblts a
much hlgher lrequency in
fhts dlsonse group.
I
I
L
ngum 11.26 Hbplotype rssoclatlon allows high-rtmlutlon gene mapplng, (a) Reprentatlon of the same subchromosomal region in
dltf~rmt lndlvidualr wlthln an ancestral population that lived several thousand yean ago, Llnes of the same color haw the $amp set of alleles
kaure of common dexent. The disease mutation (M) occurred on one ancestral chromosome. (b) After 40 generations, the original
subchromommal region has broken apart through recornblnation Into three smaller reqlons (1 on Ihr lefl, 2 In I ~ rnrddk, P ond 3 on the dghl). Each
smaller region still occurs in three different haplotypes. (c) You now collect DNA sampler from a number of rap, unrelated p-IP
with the disrar*
and from an equal number of people without the disease, and compare the frequency of each haplotype lmrn reglonr A, R, and C In the disease
and conlrol grotrps. G~nntyplng of the sampl~r will ~nnble you to cornpaw haplolype lrequencles in the wo groups. Haplotyp~ 01 rcglons not
clo5rly linked lo th~
disraqr locus wlll occur with rh~
same lrqu~ncy in both qroups. TIlp only signincant diff~r~nc~ will l,r iri the one haplotyp~
(hat encampiswr thr druwse locus (the biup haplotypp of reglon R); it ha1 a nrrlch Iilgh~r frequency fiere 100%) in !hc d~scasc group than in !he
ronlrnl group.
(8) Formetlan d haplotypas over evolutlonery tlme
@Ib)
Compsrlson of two final haplatypH wlVl s mpllatlan of rive SHPu spread over 5000 bnwi palm
Haplolym 1 .r ACGOACTOAC -
Hawtype2 II ACTcMW -
- - -
CCTTAC~TRI TACTA~CAI ATATCGUTM
I I 1 I I
CCATACGTrCl -
TACIAGCICAT -
At;ATfXKCM -
f@m 11.25 Haplotypes are fonncd by rcqucntlel mutatknr In r srndl g-mk region. (a) Tw~
pdhwtlp 01 di~~rq~nc~ from an
or1qinal5 kb chmowmal rqian (d~rphyd In Ihr mMdlr of rh~ fwo dwrgmc palfiwoyJ). Alonq one pathway, Iwo mutation+ accumulate; alonq
tlir ~mmd parhway, thrw rnulalbns ~ccumulat~. (b) Thr sun+mng dmudant chmmmomm wpmrnt twtl distinct haplolps Illat dllfer at IIW
rrparalc SNP focl.
-