Genetic polymorphism of goat CSN1S1 and CSN1S2 genes and their impact on milk composition

I

I

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Universitat Autònoma

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_

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of Medicine)

Dr. Armand

Srlnchez

(Dep.

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'.; l

t

I

Genetic

polymorphism

of goat

CSNlSl anil

CsN1s2 genes

and

their

impact on

milk

composition

Cosenza G.,

Pauciullo.d,

Ramunno

L.

3i?*ffi

T:,$i§ffiffi

_ffiTH*":fff

1i$:,1"iJhfl"fr:li;

As

it

is well known,

six

main proteins are present in ruminants

milk

four caseins

(osl,

crs2,

₀

e

k)

atld two main whey protein, _{B-lactoglobulin} and o-lactoalbumin. In ruminants the four caseins repiesent about the 80%

of milk

proteins. Three (4s1, _{B and}

os2)

of

the

four

caseins are sensitive

to

calcium

precipitation,

show

similar

molecular

weights (around

24

tDa),

promoter regions, leader peptide

§€quences and locations

of

the major

phosphorylation

site.

These data support

ttr

hypothesis

of

a

common evolutionary origrn of these genes _{from the duplications of a unique ancestral g"oà}

Jf 1.

At

the presen! the

genomic

orgauzation and the nucleotide sequences

of

these g-enes

_-L

koo*r.

These genes _{have been mapped}

_in

_{the order}

_C§i//51,

_{CSN2, CSNI§2 and C5NJ}

_n

_alSO

_kb

_(kilobase)

DNA

region of chromosome 6

in

cattle and goat. In goat the

CSI/I§/

and CSN2 genes are conìergently

franscribed at only 12 kb apart

_ll;21.

The goat CSN,I^S/ gene extends over 16.7 kb including 1138 bp

of

exonic regions and about 15.7

kb

of intonic

regions. The

main

feature

of

the goat

C§l/1§/

gene

is

the

extremély

split

architecture.

It

contains 19 exons rangng

in

size fuom24 to 385 bp and 18 introns from 90

to

16g5 Up _f:1.

The goat CSNISI _{gene represents, since many years, an excellent model for demonstrating that the}

major part of the

variability

observed in the cr,sl-casein content in the goat

milk

is due to the presence

of

autosomal alleles at a single skuctural locus (CSN7S7). So far, atleast 17 alleles have been identified,

which are associated to different levels

of

osl-casein expression

in

the

milk. A

first group of alleles are

related

to

a

nomtal content

of

gsl-casein,

_whereas

_I

_and

_E

_{alleles are associated}

_to

_{an intermediate} content, and D, F and G alleles are related to a

low

level

of

crsl-casein

in

the

milk.

Alleles

Csi//S/

N,

0l

and

02

are'null'

alleles and have been associated

with

the apparent absence

of

crsl-casein

in

the

milk

_[

3;4;5;6].

Most

of

the

mutational events responsible

for

the formation

of

such alleles have been already

identified: particularly, the alleles associate

to

a normal content

of

this

casein fraction have originated

from single nucleotide substitutions

_[a;5;6].

_{While the molecular event characterizing the}

_I

_{allelJ is not}

known _[4J, the E allele is characterized from an insertion

of

a

DNA

segment

(LINE,Lrrg

Interspersed

Nuclear Elemen! 457 nucleotides long) which took place inside of the

Dtr

exon _[7].

Regarding the alleles related to a

low

level

of

osl-casein in the goat

milk,

for the cr,sl-Cn

D

and G

alleles, were observed

mRNA

characterized by the out-splicing

of

tfre exors

i

and 4, respectivefy

_i3t.

For the

G

allele,

oDNA

sequencing revealed that exon

a is

skipped during the course o1.

pr.-rnùa

processing. The mutational event responsible for the outsplicing

of

exon

r"quro*,

is a

tansition

(G

-+

A)

occuning in the

5'

splice site consensus sequence _t5]-

_At

this moment,

iì

is unknown the molecular

ovcnt responsible

ofthis

erroneous splicing in D allele

The

F

allele is, instead, characterized from a deletion

of

the 23'd nucleotide

of

the 96 exon and the insertion

of l1

bp and 3 bp

in

the subsequent intron. By means

of

Northern blot analysis the amount

of

osl

casein

mRNA

transcribed

from

F

allele

was estimated

to

be at

least

6 time

lower than

ttrat transcribed from

A

allele

_[9].

_{Furthermore, the F allele was shown to yield multiple alternatively spliced} hanscripts (at least 9), among

which

the most representative

_mRNA

population

is

characterized Uy

tfr"

alternative skipping of exon 9, 10 and

l1

and responsible as consequence, for

the

synthesis of a

fofo

of

crsl casein deprived of 37 aa _{[3; 9].}

The 01 allele, the true

null

allele, is characterized from the deletion

of

a

DNA

segment of nearly g.5

kb (starting from the 181 nucleotide

of

the

intron

12, and including the last 7 exons of the genel jtO1,

while a large insertion, so far uncharacteize4 is the mutational

"r.Jr.rponsible

of the 02

alje

fjt.

-ILecently, in a goat population _{reared in} the Naples province, a new allele at the this locus (named

N)

have been _{identified, being associated to an apparent lack}

ofsynthesis ofgsl-casein [3].

Sequence data rurd typing results show that

the

CSM/^§1

_N

allele is characterized,

like

to

the CSN1S1

F

allele

by

the

'deletion

of

cltosine

at the

nucleotide

23 of

the

exon

9.

The

cytosine deletion

is

resulting

_in

one-nucleotide frameshift

and

detennines

a

premafure stop

codon

ii

"*or,

12 t3l.

The presenée

in

the

("^§ff/§/

_N

_{allele of a premature terrnination codon}

eTò)

might be responsibll ior the apparent lack

of

trs

I

casein synthesis.

_The

_correlation

_between

_the

_presence

_of

_{a PTC}

_and

_the

absence

_of

protein

eynthesis has been also found

in

other eukaryotic genes

_[

1] and,

in

particular, _{at the}

_loci

_ofother

_two

Ca'acn§itiv._rT9i*

of

goat

g;s2 _{[12] and}

B

[B; u].

However,

N

allele is characterized by the absence, rcontrary

to

F allele,

of

the

insertion

of

lt

and

3

bp

in

the subsequent

intron

_[3].

The molecular data iuveilable, in would allow to hypothesize that ttre

CSlnsr

N

allele

èodd

Ue _{originateA by an}

_interallelic

j:rccombination event _[3].

;.-*:ly:::?{

T,tTt

of

Quantitative Real Time PCR shows that the amount of mRNA ranscribed

by

il1t-:.Yj_t1-L1l,:

jt

up.nT","tly.t"

33Yo of .tbat hanscribed

by the cst/1^sl

F auele. comparison

of

'1Pts Rroauced

by the

N

and

F

alleles shows a remarkablé

variabitity

in

alternative

splicing

(at

12 populations

of

CSNIS1

N

mRNA).

Particularly,

_for

_the

_F

_{allele u}

_n4rc,

_{ratio was observed}

:omparcd to the

N

allele

in

the amount of uanscrip* characterized by

outsplicirfof

exons 9,

l0

and

l

l

i:*jlll

_lgoat

_C§il/§/

::"?ty:)

_gene

t3l.

It,1t possible to

hypotheiin

Ptthe

observed differenles in the expression

of

could be the direct consequence of more elaborated systems of

grr"

regplation.

3,:f::

:*:1:.T:,,",

ry]f:Thism

onmirk

yield. andcomposition, miceue sfucture, renneting

rpatier and cheese yield have

6".n

lfosletrghly smàied _{in different breeds. Results can be summarised}

Ilj,"t:1;tlÌ

y-,Iry:enc:s

exist

among genotypes.with respect

to milk

yields;

_e)

_it

shows a

tgnlflcant offect on the diameter

of

the miceltes _{andòn their calcium content which are lower}

_{in milks}

AA";

(3) goat

milk

*t

ttn

levels

of

asl

casein has a better

milk

composition, including total solids,

ht

proteLn, casein _{phosphorus and lower pH than milks}

_with

_low

_levels

_of

_{qsl-casein; in particular,}

the

:fnflYITTl_E^-1ry*

to,Tfrove

1il{

comnosilol

over

null

variant

milk

composition

(4)

goat

ffiffi'*ff;;

:jff:r,m::ill

flT.{o1

genom.' .oi"titot

a

_'ia

1*o"e,

il;il;h;*,

betrer renneting/vrlvr rvulwtulÉ

ior

(faster coagulation

and

firmer

cwd) than

"intermediate"

Ld

..null,,

grrotl.p"r-,

ffi;;;

bettcr properties than uweak" _{genotypesi and (6) cheese yields}

_of

_{different;.;;tJrp",}

are ranked

in thommo lvay a§ for renneting

propertiesf(f

cheeses made

with milk

from these _{genotypes have less}

vp§lf:11,11:.T-lf

those

frq

Y$'gr*typer,

dye to different

atry

u.ii

_{p.ofiles [review}

r5].

Ooat

milk

proteins have many significant diffeÉnces _{in their amino}

_aià

cr.poril;';;;

_àà

_,i"rt

othor mammalian species, especially in relative proportions

_of

_{the various}

_miik

_{proteins and}

_{in their}

ic

polymorphisms

_{The major}

_protein

_in

_cow

_milk

_is

_{osl-casein, but}

_goat

milk

may differ

ically _{by having either none or much. This in turn indicates and may}

_{.*pruioiigoificant}

differences

o §ow

milk

_in

digestion

by

infants and patients

_{[16], which}

_{taditionally riru.}

_{been explained}

_by

_the

homogonized" nature of goat

milk

fat.

Bevilacq.ua _ot

al.llTl

_{noted that "conhadictory results have been reported on the}

use

of

goat

milk in

[,

i:[fl:pLlt

w11 sugg^este9

tu,

this could be

{ue

to

this

"high

genetic polymorphism

of

goat

rllk

protcins".

The

authors

_{found that}

guinea

_{pigs fed}

goat

_milk

_with

_low

;;il;;ff;

|":il'fll|.

f:

:IT*..'.1"

B.j"'*9sloryF-tnan

animar'

r"a

*ia

goat

milk

containing higher

cr,st-Pin'They

suggesrcd that the digestion

of

B -lactoglobulin was enhanced in the relative absence

of

*-l -casein.

To

date,

the

7

alleles

_{identified at this}

_locus

_{would seem}

_to

_be

_associated

_with

three

different

ixpression levels: whereas CSNLS2A,B, C, E and _{F alleles are associated}

_with

_{a normal content}

_of

as2-In

_{recent years a remarkable ggnetic polymorphism has been revealed also at}

_the

CSNIS2 locus rn

vuf f tp- avva L,.l

fne

cs{lsf

g:ne.org}zationjt

y..y

simitar to that

of

genes coding

for

the other

two

calcium

,id;à;-ii;il;d;;;'[i

^ a<a -.-^1^^r:J-- rt 6r

7

I

t

I

I

I

t

I I

I

l

I I I I É I c I § E E È

casein

_{U9;20;21;22;231}

the CSNIS2D allele is associated

with

an intermediate amount of this casein

fraction [23].

The CSil/,S20 allele

is

a

"null"

allele being associated

to

the

apparent absence

of

os2-casein in

milk

_[12].

At

molecular

level,

the CSNI§2A,

B,

C,

E

and

F

alleles

differ

in

point

mutations

and,

as

a

oonsQquence,

in

amino

acid

substitutions

at

the protein

_{level [21; 22; 23].}

The

C,S1//^§2D

allele

is characterized

by a

106-bp deletion,

involving

the last 11 bp

of

the

exon

11 and the

first

95 bp

of

the subsequent

inton

_[23].

The mutation

that

characterizes

the

null

allele

is

a

transition

(G-+A)

at nucleotide 80 of the exon 11. This mutation is responsible

of

the formation

of

a premature stop-codon and,

of

consequence,

for

the apparent lack

of

os2 casein synthesis _[12].

Dot blot

analyses showed that

the level

of

C,Sil/^S2

mRNA

of

mammary gland cells from the CSi//.S2 010 goat

is

about 10%

of

the

normal value [12]. Likewise

for

the

goat C§l{1,S1 gene, the analysis

of

the length

of

cloned RT-PCR

ftagments, identified 2 mRNA populations for

A

allele, 3 for the

D

allele

_[23]

and at least 6

for

0 allele

1241,

Alleles

associated

with

a

null

amount

of

protein have been

found

for

the

an other goat calcium

raruitive

casein fractior5 _B casein

_fla;251.

Therefore, at presen! Capra hircus is the

only

species

for

whloh a null allele is available for each of these proteins.

In conclusion, by merns

of

selection based on parent genotyping at the

DNA

level

it will

be possible

b

ohain

goat populations producing

milk

characterised

by

the absence

of

either gsl-casein,

or

os2-ortÉin _{or É-casein. Such}

milk

could be usefrrl

for

qpecific technological processes

of

kansforrnation or

&r

rpocific

ntrtritional

or

dietary purposes

in

order

to

attenuate

the

nègative consequence

of

some

ugtrbolic

deficiencies and allergies and to contribute to the prevention of some diseases

Coflsidering

the

remarkable quantitative polymorphism characterizing

the

main protein

fractions

Ming

gene, some goat

_{milk §pes}

are more similar to the woman

milk

and, thereforè, could be used,

qdù

_{botter results, in the human feeding. In fact, the woman}

_milk

_{possess particular}

characteristics.

It

is

k§ldtrg in

the p-lactoglobulin

fraction

and

it

is

characterized

by

a

general

low

content

in

casein,

in

pldaular,_crs2

casein absence

and

crsl

casein haces,

similar

to

the

milk

produced

by

goats

with

ffre§pe

CSNIS2 010.

Tht

*ilk

n-rodyced.by goats homozigotes for C§i//,S2 0 allele,

b"irg

characterized by the absence

of

:UOh protein fraction, is similar

in

composition to the woman

milk

an{therefore,

it

couid, perhaps,

find

Frofi!

employment in the feeding

of

the newborn,

limiting

intolerance phenomena to this

specific'milk

protcin fraction.

References

l.

Rijnkels,

M.,2002.

Multispeoie comparison

of

the

casein gene

_loei

and evolution

of

casein gene family. J. Mammary Gland Biol.

7,327145.

2.

Leroux

and

Martiru

1996.

The

caprine

asl-

and

p-casein

_are

_l2-Kb

_apart

_and convergently tanscribed. Anim. Genet.,27 (suppl.

Z),93.

3.

Ramunno

L.,

Cosenza G., Rando

A,

Pauciullo

A, Illario

R,

Gallo

D,

Di

Berardino D.,

Masina

P.,

2005. Comparative analysis

of

gene sequence

of

goat C,SN1S1

F

and

N

alleles and characterization

of

c§l/1§/

tanscript variants in mammaly gland.

Gene,34s

Q),289-299.

4,

ehianesc, _-I..,

Fgraud,

P,, Garrg, G., Mauue!-lg, R., Addeo,

F,,1997.

osaurergp of t&rce

novel

alpha

sl-casein variants

in

goat

mik

Milk

Protein Polynorphism

FIL-IDF

Palmerston North, New Zeland, 259-267 .

5.

Martin,

P., ollivier-Bousque!

M.,

Grosclaude,

F'.,

1999. Genetic polymorphism

_of

casein: a tool too investigate casein micelle organization. Int. Doiry J.

§, t63-:,7t.

6.

Bevilacqu4

c.,

Ferranti, P., Garro, G.,

veltri, c.,

Lagonigro,

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c.,

pieholà,8.,

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F.,

Chianese,

_L.,

Martin,

P.,

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recombination is probably responsible

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the occrurence

of

a new alpha(sl)-casein variant found

in

the goat species. Eur. J. Biochem.269

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1303.

Jansà Pérez

M.J., Leroux C.,

Sanchez Bonastre

A., Martin

p.,

lgg4.

occurrence

of

a

LINE

sequence in the

3'UTR

of the goat osl-casein E encocling allele associated

with

a reduced protein synthesis level, Gene 147,177-179.

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F.,

Ricordeau

G., Martin

P., Remuef

f.,

vassal

L.,

Bouillon

J.,

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goat osl-casein

transcripts. structural organization of the relevant gene. J.

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G.,

Illario

R,

Rando

A.,

Di

Gregorio

p.,

Masina

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Ramunno

L.,2003.

Molecular characteization

of

the goat CSNIS/OI allele.

Journal

of

Dairy

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ll-Losson, R.,

Lacroute,

F.,

1979. Interference

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nonsense mutations

with

eukaryotic messengerRNA stability. Proc. Natl. Acad. scr.

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L.,

Longobardi

E.,

Pappalardo

M.,

Rando

A.,

Di

Gregorio

p.,

Cosenza G.,

Mariani P., Pastore

N.,

Masina

P.,

2001.

An

allele associated

with

a non

detectable

amount of os2 casein in goat milk. Anim. Genet. 32

(t),19-26.

13. Ramunno

L.,

Mariani P.,

Pappalardo

M.,

Rando

A.,

capuano

M.

Di

Gregorio p.,

Cosenza G., 1995.

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gene ad effetto maggiore sul contenuto

di

_{caseina B} nel latte

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capftL Atti

fl

Cong. Naz. A.S.P.A., Grado (Go), 185-1g6.

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M. A.,

Printz C., Medraoo J. F., Mercier J.C.,

lggg.

Asingle nucleotide deletion resulting

in

a premature stop codon

is

associated

with

marked reduction

of

hanscripts from a goat beta-casein

null

allele. Animal

Genetics,l[,4444Sl.

15. Serradilla J.M.,

20!]

The goat alphasl-casein gene:

A

paradign of

the use

of

a major

gene to improve

milk

quality? In Gabifla D. (ed.), Sanna S. (ed.). Breeding programmes

for

improving

the quality

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safety

of

products. New

traits, tooTi

rùles

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or ganizat ion? Tarugoza:

CIFIEAM-IAI\M,

99 ^t 06.

16.

Mach P.8.,

1953.

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preliminary nutrition study

of

the value

of

goat

milk

in the diet

_of

children. Amer. Goat soc.

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year

_Book

_{lgs2-lg53,ll2-132.}

17. Bevilacqua C.,

Martin P.,

Candalh

c.,

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piot

M.,

Roucayrol

A.M., pilla

F.,

Heyman

M.,

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milk of

defective

alpha(sl)-casein

genotype decreases

intestinal and systemic sensitization

to

HaJactoglobulin in

guinea pigs.

i

Dairy

Res.

69,217-227.

18. Groenen

M.A.M.,

Dijkhof

RJ.A.,

verstege

A.J.M.,

van

der poel

J.J.,

1993. The complete §equcnce of _{the gene encoding bovine qs2-casein. Gene}

_l23,lg7-g3.

19. Boulanger

A.,

Grosclaude F., Mahè M.F., 1984. Polymorphisme des caseines crsl et crs2

de la chèwe (Capra hircus). Genet. Sel. Evol.

16,lS7-175.

20. Grosclaude

F.,

Mahè

M.F.,

Brignon

G.,

Di

stasio

L.,

Jeunet

R,

19g7.

A

Mendelian polymorphism underlying quantitative variations

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7

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