INVERSA ELECTRÒNICA

I

E n d o th e lin R e c e p to r A E n d o th e lin R e c e p to r B

F ig u re 2 .4 T w o -sta g e s y n t h e s is o f t h e e n d o th e lin -1 p e p tid e . T h e y e llo w h ig h lig h te d s e q u e n c e is t h e b ig e n d o th e lin 1 p e p tid e . T h e g r e e n h ig h lig h te d s e q u e n c e s are ta r g e t s i t e s for a c t io n o f t h e p r o te a s e s .

m o u se k n o c k -o u t w ork th a t th e E T -1 /E C E -1 /E T A p a th w a y is cru c ial

for n o rm a l d ev elo p m en t of th e first a n d seco n d b ra n c h ia l a rc h e s .

B a rn i et al (1998) h av e h y p o th e sis e d th a t ET-1 p ro m o te s

b ra n c h ia l a rc h d ev elo p m en t d u rin g w eeks 11-12 of h u m a n

em bryological developm ent, coinciding w ith d ev elo p m en t of th e face.

It is en v isag ed th a t th e a ctio n of ET-1, m ed iate d by ETA a n d EC E-1,

to g e th e r w ith th e different p a tte r n s of e x p re ssio n in d ifferen t cell

c o m p a rtm e n ts lea d s to a differential re s p o n se to cell p ro liferatio n a n d

b o n e fo rm atio n . T he p h e n o ty p e of ET-1 - / - m ice h a s c o n sid e ra b le

sim ilarity to m a n d ib u la r a b n o rm a litie s sh o w n in h u m a n sy n d ro m e s

w h ere th e first b ra n c h ia l a rc h is affected s u c h a s T re a c h e r C ollins

(OMIM 154500) a n d Pierre R obin sy n d ro m e (OMIM 2 6 1 8 0 0 ). T he fact

th a t b o th th e face a n d th e h e a r t a re affected h ig h lig h ts sim ilarities

w ith th e card io -facial c o n d itio n s collectively g ro u p ed a s CATCH 22

(Wilson et al 1993).

2 .6 . M ethods o f gen etic analysis

M apping s tu d ie s a n d id en tificatio n of m u ta tio n s in g e n es th a t

c a u s e cran io facial d efects e n a b le s som e u n d e rs ta n d in g of th e

p a th w a y s involved in th e d ev elo p m en t of th e face a n d m ay h e lp to

identify c a n d id a te g en es re sp o n sib le for n o rm a l p e rs o n to p e rs o n

v a ria tio n . T h ese a p p ro a c h e s h av e b e en em ployed to identify g en es

resp o n sib le for b o th com plex a n d sim ple pathology.

th o u sa n d o u t of the total 3000 million nucleotides, m ean in g an

average of 3 million differences betw een any two individuals.

Therefore w ith o u t th e recen t rapid progress m ade in gene

identification, function an d expression stu d ies along with progress

m ade in sequencing an d single nucleotide polym orphism (SNP)

identification, the process of identifying genes responsible for h u m a n

variation in facial featu res would have been an im possibly difficult

ta s k to u n d e rta k e.

Complex genetics focuses on individual variation in co n tin u o u s

traits. These are graded series of phenotypes from one extrem e to

a n o th e r w ith no clearly d istin g u ish ab le su b se ts a n d w here th ere are

often elem ents of gene-environm ent an d gene-gene interaction. These

tra its include height, weight, an d IQ. Complex tra its are often

m ultifactoral; th e phenotype being d ep en d en t on environm ental a n d

n u m e ro u s genetic factors. Also, in c o n tra st to tra its w hich follow a

sim ple M endelian p a tte rn of in h eritan ce the genes c o n trib u tin g to

complex tra its have relatively sm all effects, a n d act in concert: as

su c h they c an n o t be easily identified th ro u g h segregation analysis.

Very few facial features have been do cu m en ted as being

in h erited in a sim ple m an n e r following a M endelian p a tte rn of

in h eritan ce. F u rth erm o re, it is likely th a t the genetic m ech an ism s

underlying in h eritan ce of norm al facial featuresCîCcomplex in order to

a cco u n t for th e im m ense p erso n to perso n variability. It is a ssu m e d

c o m b in a tio n s of g en es t h a t a re su b je c t to th e sa m e law s of

tra n s m is s io n a s sim ple tr a it loci. T herefore th ro u g h th e a n a ly sis of

large fam ilies, a n d b re a k in g dow n th e com plexity of p h e n o ty p e s in to

sim ple c o m p o n e n t p a rts , th e effect of e a c h c o n trib u to ry gene m ay be

a sc e rta in e d . For exam ple, if th e face w ere to be c ateg o rise d into 20

po ly m o rp h ic fe a tu re s a n d e a c h of th e 20 fe a tu re s h a d 3 p h e n o ty p e s,

th is alo n e w ould a c c o u n t for or 3 4 8 6 7 8 4 4 0 1 facial c o m b in atio n s.

T his sim ple c a lc u la tio n ig n o res v ariab ility d u e to o th e r fac to rs s u c h

a s age, e th n ic b a c k g ro u n d , sex, a n d w eight.

Practical consid era tio n s

W hen try in g to identify g e n es resp o n sib le for p a rtic u la r p h e n o ty p e s th e re a re b ro ad ly sp e ak in g two stra te g ie s th a t c an be u se d , linkage or a sso c ia tio n a n a ly sis.

L inkage a n a lysis-h n ksig e looks a t p h y sical re la tio n sh ip s betw een loci. The

u n d e rly in g aim is to see how often a m a rk e r lo cu s a n d a tra it lo cu s is s e p a ra te d by m eiotic reco m b in atio n . The n u m b e r of inform ative m eio ses re q u ire d to d e te c t linkage is d e p e n d e n t on th e reco m b in atio n rate: w ith no re c o m b in a n ts it is

e stim a te d t h a t 10 m eio ses a re sufficient to d e te c t linkage w h e re a s w ith a

re c o m b in a tio n fractio n of 0 .3 , 85 w ould be req u ire d in o rd er to get th e sa m e level of significance. The a p p ro a c h d e p e n d s on collecting fam ily d a ta from large

p ed ig rees o r n u m e ro u s sm all p ed ig rees a n d looking for linkage b etw een th e sco red p h e n o ty p e a n d know n g en etic m a rk e rs. The efficacy w ith w h ich linkage c a n be u s e d to m a p tr a its is v ariab le a n d relies in p a rt u p o n th e type of m a rk e rs u s e d a n d how d en sely th ey a re sp a ce d th ro u g h o u t th e genom e (K ruglyak 1999; C ollins e t al 1999; W ang et al 1998; S ch afer & H aw kins 1998).

P ro b lem s a sso c ia te d w ith linkage in clu d e th e n eed for inform ative m eio ses a n d co m p lete specification of a g enetic m odel of th e tra it being m ap p e d , in clu d in g m o d es of in h e rita n c e , seg reg atio n d a ta , a n d e stim a te s of gene freq u e n cie s a n d p e n e tra n c e [M ueller a n d Cook 1996; L athrop, Terwilliger & W eeks 1996; S pence a n d Hodge 1996; W right C a ro th e rs & P ira stu 1999; W ang 2000]. O th e r p ro b lem s in clu d e lo c u s h etero g en eity a n d false re c o m b in a n ts [W hittem ore a n d H alp ern 2001]. M odel-free m e th o d s of lin k ag e a n a ly sis have also b een developed,

in clu d in g th e im p o rta n t sib -p a ir a n a ly sis a p p ro a c h , w here efforts a re fo cu sed on looking a t ch ro m o so m e se g m e n ts s h a re d by affected in d iv id u als. In th is m eth o d a lim iting fe a tu re is th e n eed to collect large en o u g h n u m b e rs of sib p a irs to achieve s ta tis tic a l significance, especially w here genetic h etero g en eity is a

problem . O verall, linkage a n a ly sis h a s b een th e m ajo r m eth o d for identifying loci th a t d e te rm in e single gene d is o rd e rs or c o n d itio n s in w hich relatively few g en es

A sso c ia tio n a n a lysis- allelic asso c iatio n s tu d ie s look a t th e re la tio n s h ip s betw een alleles, e ith e r in affected fam ilies or a c ro ss p o p u la tio n s of affected a n d u n a ffe cte d in d iv id u a ls, to see to w h a t e x te n t a n allele a t a p a rtic u la r m a rk e r lo cu s is

a s s o c ia te d m ore freq u en tly th a n ran d o m ex p ectatio n w ith a tra it. A sso ciatio n b etw een a n allele a n d a tra it c a n a rise e ith e r b e c a u se th e allele c a u s e s th e tra it d irectly o r is very closely linked to a n allele th a t does. Allelic a sso c ia tio n a n a ly sis is a n e sse n tia l tool in d e te rm in in g th e genetic b a s is of com plex d is o r d e r s /tr a its w h e re linkage a n a ly sis is difficult. A m ajo r p ro b lem w ith a sso c ia tio n s tu d ie s is p o p u la tio n stra tific a tio n , w h ich a ris e s largely from e th n ic diversity, b u t age a n d g e n d e r m ay also ex ert a n influence. T h u s in c a s e /c o n tro l s tu d ie s it is ex trem ely im p o rta n t to select a su ita b ly u n b ia s e d co n tro l cohort. [Schork et al 2001].

A n o th e r p ro b lem w ith a sso c ia tio n is th a t th e sta tistic a l a n a ly sis is often n o t su ita b ly s trin g e n t w ith referen ce to the n u m b e r of q u e stio n s a sk e d , s u c h a s the n u m b e r of loci te ste d . T h u s th e B onferroni co rrectio n , w hich ta k e s th is in to

a c c o u n t, sh o u ld alw ay s be ap p lied (p= 0 .0 5 / n w here n is th e n u m b e r of q u e s tio n s a sk ed ). D ue to th e p ro b lem of finding su ita b le co n tro ls, a sso c ia tio n m e th o d s w ith in te rn a l c o n tro ls a re now often u se d (L ander a n d S c h o rk 1994). For ex am p le, TDT (tra n sm iss io n d isto rtio n test) u s e s n u c le a r fam ilies w ith one or m ore affected o ffsp rin g a n d o n e p a re n t h etero zy g o u s a t th e m a rk e r lo cu s u n d e r in v estig atio n . T h is te s t c o m p a re s th e freq u en cy w ith w hich th e p a re n ta l alleles are tra n s m itte d

to offsp rin g w ith th e tr a it/d is o r d e r . D eviations from th e 5 0 :5 0 ratio of

tra n s m is s io n in d ic a te a n a sso c ia tio n (Spielm an et al 1993). S u c h m e th o d s have