IDENTIFICATION !

OF GENES !
RESPONSIBLE FOR"
MONOGENIC !
DISEASES!

P2 family

I-1 I-2

II-1 II-2 II-3 II-4 II-5 II-6 II-7 II-8 II-10 II-11 II-12 II-13 II-14

Retinitis pigmentosa

Down syndrome
Lod scores between ARRP and markers on 2q in family P2

Marker!! Recombination fraction (!)! ! ! ! ! ! !

! ! ! ! ! !
! ! 0.00! 0.01! 0.05! 0.1! 0.2! 0.3! 0.4! Zmax ! !max
! ! ! ! ! ! ! ! !
D2S148! -"! 1.53! 1.95! 1.91! 1.51! 0.95! 0.34! 1.96! 0.06
D2S364! 4.03! 3.93! 3.55! 3.07! 2.15! 1.26! 0.43! 4.02! 0.00
D2S350! 2.19! 2.14! 1.93! 1.68! 1.19! 0.72! 0.27! 2.19! 0.00
D2S318! 1.99! 1.95! 1.76! 1.54! 1.10! 0.68! 0.25! 1.99! 0.00
D2S118! 4.12! 4.02! 3.63! 3.14! 2.19! 1.28! 0.44! 4.12! 0.00
D2S389! 4.12! 4.02! 3.63! 3.15! 2.20! 1.29! 0.44! 4.12! 0.00
D2S161! 0.31! 0.41! 0.60! 0.66! 0.58! 0.38! 0.13! 0.66! 0.11
D2S117! -"! 0.77! 1.45! 1.56! 1.28! 0.79! 0.27! 1.56! 0.09
! ! ! ! ! ! ! ! !
 * Now replaced by the
“positional candidate
approach”

Theta (!): recombination fraction

Phase: set of alleles (of different loci) inherited from the same !
parent !
Let’s suppose that “D” and “M” are the paternal alleles and !
that “d” and “m” are the maternal alleles. !
Theta (!): recombination fraction

If two loci are in different chromosomes, they would segregate !

independently to form the gametes. !
The proportion (or fraction) of recombinants (Dm o dM) will be !
50%. Thus, it will be ! = 0.5.!

Theta (!): recombination fraction

If the two loci are in the same chromosome, and at a very short!
distance, they would go together to the same gamete. !
The recombination proportion or fraction (Dm o dM) will be !
practically zero. Thus, it will be ! = 0.!
Theta (!): recombination fraction

If the two loci are in the same chromosome, at a certain!

distance, a meiotic recombination may take place between them. !

The recombination proportion or fraction (Dm o dM) will not be !

null. If the distance between the loci is relatively small, !
it will be 0 <! < 0.5.!

Theta (!) and cM

As the distance between the loci become!
larger, ! become larger, because of the!
increase in the recombination probability.!

! = 0.5! 190 cM! Thus, ! corresponds to a distance. !

But this is true only for short distances, !

because from some point onwards,!
! will be always 0.5.!
! = 0.5! 120 cM! A MAP FUNCTION transforms!
! in cM.!
! = 0.5! 78 cM!
1 cM is a distance between two loci!
in a chromosome for which 1% !
! = 0.5! 60 cM!
recombination is expected.!
! = 0.2! 21 cM!
Now we can see distances in cM along!
! = 0.01! 1 cM! the chromosome. Human chromosomes !
are between 100 and 300-cM long. !
 LINKAGE ANALYSIS

Aim: to determine if two loci are linked or not.!

That means, whether ! is SIGNIFICANTLY < 0.5!

We normally perform the analysis between a MARKER

for which we know the location and a disease LOCUS. So,
the result indicates the genomic region for the LOCUS
(close to the marker for which we found a ! value < 0.5)!

It can be also used to estimate the distance between the

LOCUS and the MARKER (in terms of !) .!

LINKAGE ANALYSIS

First, we should count the number of recombinant a non-

recombinant individuals in the pedigree.!

Then, we need to calculate (for different values of !) how

more probable is that the results are due to the fact that
the two loci are linked (at that value of !) than they are
due to chance.!

The LOD SCORE (Z) is the logarithm of the probability

quotient:!
Z = log Probability of the data if ! = X!
Probability of the data if ! = 0.5 !
 LINKAGE ANALYSIS

A LOD SCORE " 3 is generally taken to indicate that two

loci are linked. #
It is an arbitrary value, similar to p < 0.05 o p < 0.001.!

In fact, as it is a logarithm, it could be interpreted that the

value of 3 corresponds to p < 0.001 (however, there are
different opinions)!

A LOD SCORE $ 2 is generally taken as a proof of

exclusion: the locus is NOT linked to the marker (at least,
for the value of ! for which the result below -2 was
obtained)!

LINKAGE ANALYSIS
How to establish the phase.!
I cannot establish phases
Dd! dd!
12! 22! for the mother.!
I know what each child
inherited from the mother, but I
dd! Dd! don’t know if the 3 of them are
11! 12! R or if the 3 are NR.!
There are samples from the los
grandparents!!
Dd! dd! Dd! The grandfather does not help.!
11! 12! 11!

But the grandmother does:!

D goes with 1.!
 LINKAGE ANALYSIS
Example with known phase. For ! = 0.!
Only used to !
D d! d d! Only maternal meioses are
establish! 1 2! 2 2!
phases! taken into account.!

D1 (probability = 1/2)!
d d! D d!
1 !1! 1 2!
d2 (probability = 1/2)!
(No other gametes are possible !
because we are under the ! = 0 !
hypothesis)!
d D! d d! d D!
1 1! 1 2! 1 1! The joined probability for these!
NR! NR! NR! data under ! = 0:!
Prob.! 1/2! 1/2! 1/2! 1/2 x 1/2 x 1/2 = 1/8!

LINKAGE ANALYSIS
We need to calculate the probability for ! = 0.5.!

Only used to ! Possible gametes :!

D d! d d!
establish! 1 2! 2 2!
phases! D1 (probability = 1/4)!
d2 (probability = 1/4)!
D2 (probability = 1/4)!
d d! D d!
1 !1! 1 2! d1 (probability = 1/4)!
(! = 0.5 means that it is equally!
probable that a recombination !
occurs than it doesn’t)!
d D! d d! d D!
1 1! 1 2! 1 1! Joined probability for these!
data under the ! = 0.5 hypothesis:!
NR! NR! NR!
Prob.! 1/4! 1/4! 1/4! 1/4 x 1/4 x 1/4 = 1/64!
 LINNKAGE ANALYSIS
We said that LOD SCORE (Z) was the logarithm of the
probability quotient:!
Z = log Probability of the data if ! = X!
Probability of the data if ! = 0.5 !

In our case!
Z = log Probability of the data if ! = 0 = log 1/8!
Probability of the data if ! = 0.5 1/64!

Z = log 64 = log 8 = 0.903! Since Z < 3, I cannot

8! be sure that the locus
and the marker are
linked.!

LINKAGE ANALYSIS
Known phase. For ! % 0. For example, ! = 0.1!

d d! Possible gametes:!
2 2!

I D1; prob. = 1/2 x (1-! )!

d2; prob. = 1/2 x (1-! )!
d d! D d!
D2; prob. = 1/2 x !!
II! 1 !1! 1 2! d1; prob. = 1/2 x ! !

Prob. for III. 1 (and for III.2 and 4):!

III!
d D! d d! d D! d d! 1/2 x (1-! ) = 1/2 x 0.9 !
1 1! 1 2! 1 2! 1 2!

NR! NR! R! NR! Prob. for III.3:!

1/2 x ! = 1/2 x 0.1!

 LINKAGE ANALYSIS
Known phase. For ! % 0. For example, ! = 0.1!

Prob. for III. 1 (and for III.2 and 4):!

1/2 x (1-! ) = 1/2 x 0.9 !
Dd ! Prob. for III.3:!
1 2 !
1/2 x ! = 1/2 x 0.1!
Joined probability for these!
D1! !
d2 D2 ! d2!
data under the ! = 0.1 hipothesis :!
NR! NR! R! NR!

[1/2 x (1-! )] x [1/2 x (1-! )] x [1/2 x !] x [1/2 x (1-! )] =!

[1/2 x (1-! )] 3 x [1/2 x !] 1 = (1/2) 4 x (1-! )3 x !1 !

LINKAGE ANALYSIS
LOD SCORE (Z) is the logarithm of the probability
quotient:!
Z = log Probability of the data if ! = 0.1!
Probability of the data if ! = 0.5 !

Z = log (1/2) 4 x (1-! )3 x !1! = log (1-! )3 x !1 !

! (1/2) 4 x (1-0.5 )3 x (0.5)1! (1-0.5 )3 x (0.5)1 !

Z = log (0,9 )3 x (0.1)1 = log 0.0729 = log 1.1664 = 0.067

! !(0.5 )4 0.0625!

Z = log (1-! )NR x !R !

! (1-0.5 )NR x (0.5)R !
 LINKAGE ANALYSIS
If we use the formula: !
Z = log (1-! )3 x !1 !
! (1-0.5 )3 x (0.5)1 ! for different values of !: !

! "0.0 "0.1 "0.2 "0.3 "0.4"

# "! & 0.067 0.214 0.217 0.141!

0.3!
Z! 0.2! Zmax = 0.227!
0.1!
! ! ! !
0.1 0.2 0.3 0.4
!max = 0.25!
!!

LINKAGE ANALYSIS
! "0.0 "0.1 "0.2 "0.3 "0.4"
# "! & 0.067 "0.214 0.217 0.141!

What does it mean that for ! = 0, Z is ! & ?!

It means that there is, at least, one recombination. !

Z = log (1-! )3 x !1 ! As ! = 0, if there is at least one

recombination, a 0 remains in
! (1-0,5 )3 x (0,5)1 !
the numerator => log 0 = ! & !

If there are no recombinations, !0 = 1, and the numerator

will be % 0.!

If there are no recombinations, Zmax will be at ! = 0!

Lod scores between ARRP and markers on 2q in family P2

Marker!! Recombination fraction (!)! ! ! ! ! ! !

! ! ! ! ! !
! ! 0.00! 0.01! 0.05! 0.1! 0.2! 0.3! 0.4! Zmax ! !max
! ! ! ! ! ! ! ! !
D2S148! -"! 1.53! 1.95! 1.91! 1.51! 0.95! 0.34! 1.96! 0.06
D2S364! 4.03! 3.93! 3.55! 3.07! 2.15! 1.26! 0.43! 4.02! 0.00
D2S350! 2.19! 2.14! 1.93! 1.68! 1.19! 0.72! 0.27! 2.19! 0.00
D2S318! 1.99! 1.95! 1.76! 1.54! 1.10! 0.68! 0.25! 1.99! 0.00
D2S118! 4.12! 4.02! 3.63! 3.14! 2.19! 1.28! 0.44! 4.12! 0.00
D2S389! 4.12! 4.02! 3.63! 3.15! 2.20! 1.29! 0.44! 4.12! 0.00
D2S161! 0.31! 0.41! 0.60! 0.66! 0.58! 0.38! 0.13! 0.66! 0.11
D2S117! -"! 0.77! 1.45! 1.56! 1.28! 0.79! 0.27! 1.56! 0.09
! ! ! ! ! ! ! ! !

Family P2: haplotype for the 2q31-q33 region

I -1 I-2

D2S148 4 5 4 6
D2S364 1 6 1 3
D2S350 1 2 1 1
D2S318 2 3 2 2
D2S118 5 4 5 4
D2S389 8 5 8 3 II -1 II -2 II -3 II -4 II -5 II -6 II -7 II -8 II -10 II -11 II -12 II -14 II -15
D2S161 3 3 1 3 4 4 5 6 4 6 5 6 5 4 5 4 5 6 4 6 4 4 5 6 5 4 4 4 4 4
D2S117 9 4 6 7 1 1 6 1 1 3 6 3 1 1 6 1 6 3 1 3 1 1 6 1 6 1 1 1 1 1
1 1 2 1 1 1 2 1 1 1 2 1 2 1 1 1 1 1 2 1 2 1 1 1 1 1
2 2 3 2 2 2 3 2 2 2 3 2 3 2 2 2 2 2 3 2 3 2 2 2 2 2
5 5 4 5 5 4 4 4 5 5 4 5 4 4 5 4 5 5 4 5 4 5 5 5 5 5
8 8 5 8 8 3 5 3 8 8 5 8 5 3 8 3 8 8 5 8 5 8 8 8 8 8
3 1 3 1 3 3 3 3 3 1 3 1 3 3 3 3 3 1 3 1 3 1 3 1 3 1
9 6 4 6 9 7 4 7 9 6 4 6 4 7 9 7 9 6 4 6 4 6 9 6 9 7