(12) STANDARD PATENT (11) Application No. AU 2010202722 B2 (19) AUSTRALIAN PATENT OFFICE
(54) Title Juvenile hemochromatosis gene (HFE2A), expression products and uses thereof
(51) International Patent Classification(s) C12Q 1/68 (2006.01) G01N 33/50 (2006.01) C07K 14/47 (2006.01)
(21) Application No: 2010202722 (22) Date of Filing: 2010.06.29
(43) Publication Date: 2010.07.15 (43) Publication Journal Date: 2010.07.15 (44) Accepted Journal Date: 2012.12.20
(62) Divisional of: 2004231122
(71) Applicant(s) Xenon Pharmaceuticals, Inc.
(72) Inventor(s) Ludwig, Erwin H.;Papanikolaou, George;MacDonald, Marcia L.E.;Samuels, Mark E.;Franchini, Patrick;Kamboj, Rajender K.;Goldberg, Yigal Paul
(74) Agent / Attorney Spruson & Ferguson, L 35 St Martins Tower 31 Market St, SYDNEY, NSW, 2000
(56) Related Art WO 2000/073801 WO 2002/051438 GenBank Accession No: BAB26407 2010202722 29 Jun 2010 io 5
associated JUVENILE Diagnostic ameliorating small and
identification
organic Polynucleotide
with compounds,
HEMOCHROMATOSIS diseases
juvenile compounds,
of
agents of
kits
and hemochromatosis, iron
and
for polypeptide
are metabolism,
AND methods the
disclosed
treatment
USES
Abstract GENE
using sequences
and especially THEREOF
along
of
HFE2A
(HFE2A), methods
diseases
with for
in are
of HFE2A,
human of EXPRESSION also methods
utilizing
iron described.
patients metabolism, as
of these well
treating
are PRODUCTS for as
disclosed. mutations screening including
and/or
S&FRef: 739254D1
AUSTRALIA 2010
PATENTS ACT 1990
COMPLETE SPECIFICATION Jun
29
FOR A STANDARD PATENT
2010202722 Name and Address Xenon Pharmaceuticals, Inc., of 3650 Gilmore Way, of Applicant: Burnaby, British Columbia, V5G 4W8, Canada
Actual Inventor(s): Erwin H. Ludwig George Papanikolaou Marcia L.E. MacDonald Mark E. Samuels Patrick Franchini Rajender K. Kamboj Yigal Paul Goldberg
Address for Service: Spruson & Ferguson St Martins Tower Level 35 31 Market Street Sydney NSW 2000 (CCN 3710000177)
Invention Title: Juvenile hemochromatosis gene (HFE2A), expression products and uses thereof
The following statement is a full description of this invention, including the best method of performing it known to me/us:
5845c(2803893_1) fO 2010202722 29 Jun 2010 /S
’
diseases, 2003, entirety. applications www.ncbi.nlm.nih.gov/omim/ screening US/60/488,607, medical caused metabolism, and "juvenile OMIM™. “ Hemochromatosis identified ("HFE2 HFE")
to JUVENILE EXPRESSION
the At The methods 1.
”
(see ). by
disorder
least haemochromatosis"), especially on and disclosures
mutation present
Hemochromatosis
including
This OMIM the Johns
Serial
identification 4
of filed
iron-overload
called
basis using
type
patent
HEMOCHROMATOSIS invention
juvenile
BACKGROUND Number: in
18
methods No. of
a
this 1 July juvenile of Hopkins
FIELD PRODUCTS which gene
is
of 60/462,867,
clinical, application gene,
hemochromatosis, classic
),
2003,
relates
agents 235200;
of
disorders type designated
an are OF is
hemochromatosis such
including
and also THE autosomal hereby hemochromatosis biochemical,
3
OF generally useful University,
treatment.
(HFE3;
Online
Serial
ciaims known INVENTION THE labeled filed
AND
HFE expression incorporated in
INVENTION
No. the
Mendelian
OMIM to to
15
recessive
as
on priority
hemochromatosis
a USES and treatment the
60/498,458,
GENE gene (sometimes chromosome April hemochromatosis Baltimore,
field 604250), genetic (sometimes products
by associated
of
Inheritance THEREOF 2003, disorder, of
of reference (HFE2A),
US
iron diseases
filed
characteristics.
thereof, an called
6p21
Serial
metabolism provisional designated have MD, 28 autosomal therewith, which
in August in .3. “
type
for JH" of
been Man,
their
The No.
iron
the
or at is 2
2010202722 29 Jun 2010 10 30 25 20 15 5
type The encoding receptor-2 recessive chromosome and mutation and hemochromatosis. basis HFE2A therapeutic showing both This diseases cardiomyopathy more involved locus reported because are identifies itself HFE2A,
also
maps two typically 4
sexes
therapeutic of
may did Juvenile In The severe (HFE4;
Identification
and
HFE2A,
forms
thereby in
of included
in in
hepcidin a
of hypogonadotropic disorder, to
not
some
(TFR2;
iron
present cardiac equally. novel
be Roetto the agents. HFE2B,
2q32. iron
causes
1q21,
correspond OMIM of
disease
metabolism.
therapeutic.
SLC11A3
the
juvenile
hemochromatosis
as target facilitating metabolism.
therapeutic families which
antimicrobial OMIM
and et invention
While is whereas form JH
Diagnostic of respectively. frequent 606069),
clinical al.,
caused
1 other involves
can than q21
may
of hemochromatosis
604720), Am. HFE to gene
juvenile
JH
development
be hypogonadism,
as
Alternatively, symptoms
provides complications.
the in be
target typical
features. Administration an by
J.Hum.
linked
has compounds, the
used peptide, others
iron (OMIM used
The chromosomal autosomal mutation
chromosomal which a
for
(JH) accumulation,
to
hemochromatosis
to 2 hereditary present
to identification prevalent
it
treating chromosome Genet. before
604653), which identify If
diagnose
is
of
maps
differs the untreated, (HFE2) in
caused heart
dominant kits more
of invention the
the maps 64:1388-1393
underlying diseases
location male the and
to
and
hemochomotosis, location
which failure, gene
JH
potent are of age from which
7q22. by
HFE2A
to discover 1
the methods expression,
as q21 disorder, the
tentatively
mutation
relates
of chromosome encoding
of encodes
shows
well of
hereditary arrhythmias of typical agents begins . Hemochromatosis 30 disease genetic any
iron HFE2A
gene
(1999) as years.
more to
using
is
known
in
metabolism.
to
linkage early the designated for
ferroportin caused transferrin JH
hereditary
or the
diagnose
with mutation basis was is
effective
JH genetic but treating HFE2A 19q1
affects protein
and/or
in lethal gene
gene
is first
this
life JH by for
to 3. a
3
and predict onset and severity of adult hemochromatosis and to distinguish between types of iron metabolism disorders. 2012
BRIEF SUMMARY OF THE INVENTION Nov
5 In a first aspect, the present invention provides an isolated antisense 22 molecule complementary to SEQ ID NO: 2 or 3, wherein said antisense molecule
is the complement of the oligonucleotide of SEQ ID NO: 13, 15, 17, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, 51 or 53, wherein said antisense molecule modulates iron transport, across a cell membrane of a cell that transport iron.
10 In a second aspect, the present invention provides a method for inhibiting
2010202722 hemojuvelin activity in a cell, comprising contacting a cell with an isolated antisense molecule of the first aspect, wherein said antisense molecule inhibits hemojuvelin activity in said cell.
In a third aspect, the present invention provides a method for modulating 15 iron transport across the membrane of a cell, comprising contacting a cell with an isolated antisense molecule of the first aspect, wherein said antisense molecule modulates iron transport across said cell.
In a fourth aspect, the present invention provides a method for inhibiting hepcidin production in a cell, comprising contacting a cell with an isolated 20 antisense molecule of the first aspect, wherein said antisense molecule inhibits hepcidin production in said cell.
In a fifth aspect, the present invention provides an isolated polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9, wherein said polypeptide modulates iron transport across a cell membrane of a
25 cell that transports iron.
In a sixth aspect, the present invention provides an isolated fragment of SEQ ID NO: 26 or 27, wherein said polypeptide modulates iron transport across a cell membrane of a cell that transports iron.
In a seventh aspect, the present invention provides a composition when 30 used for treating a disease of iron metabolism comprising a therapeutically effective amount of the polypeptide of the fifth or sixth aspect in a pharmaceutically acceptable carrier.
(6885017) 3a
The invention relates to the discovery that juvenile hemochromatosis (hemochromatosis type 2A, or HFE2A), is caused by a mutation in a human gene 2012
found at 1 q22 having, the nucleotide sequence as set out in SEQ ID Nos. 1-9, and/or the corresponding amino acid sequences as set out in SEQ ID Nos. 10-12. Nov
5 The gene and the protein are referred to herein as HFE2A and also as 22 hemojuvelin, these words referring to the gene, the gene product and the protein
expressed therefrom, unless the context specifies otherwise. The gene has also been named HFE2, by which is meant the form JH caused by the gene at lq21. This naming protocol is not essential to the invention claimed herein.
10 Described herein is a method for identifying an agent that modulates hemojuvelin, comprising contacting a test compound with hemojuvelin and 2010202722 determining a change in hemojuvelin activity due to the compound, thereby identifying a modulator of the type being sought. The modulator may be a drug like small molecule, an antibody, an antisense nucleic acid, a ribozyme or any 15 other compound which changes the activity of the gene or protein.
Described herein is a method for identifying an agent that modulates HFE2A gene expression, comprising contacting a test compound with a polynucleotide comprising a HFE2A gene and under conditions promoting expression of said gene (i.e., conditions wherein the polynucleotide is being 20 expressed) and determining a change in expression due to the presence of the test compound. This identifies the test compound as such a modulator. In a preferred embodiment, this change in expression is detected as a change in transcription of the gene, preferably where the gene is a mammalian gene and most preferably where the gene is expressed by a cell.
(6885017) 4
Further described herein is a method for identifying an agent that modulates HFE2A gene expression, comprising contacting a test compound with a 2012
reporter gene operably linked to a promoter sequence and determining a change in
Nov expression of the reporter gene due to the test compound, thus identifying a
5 modulator of the construct. Preferably, this is modulation of transcription of said 22 reporter gene operably linked to an HFE2A promoter, most preferably a
mammalian HFE2A, such as a mouse, rat or human HFE2A promoter.
Also described herein is a method for treating and/or preventing a disorder in an animal comprising administering to an animal afflicted therewith, or at risk 10 of developing said disorder, a therapeutically effective amount of an HFE2A
2010202722 modulator. In a preferred embodiment, the HFE2A modulator exhibits modulating activity in a method of the invention, most preferably wherein said agent was first identified as an HFE2A modulator using said method and was not otherwise known to have such activity.
15 Also described herein is a method to diagnose individuals afflicted with or at risk of developing HFE2A or a related disorder comprising determining the nucleic acid sequence of the HFE2A gene in said individual wherein a mutation or polymorphism of said gene identifies said individual as an individual afflicted with or at risk of developing HFE2A or a related disorder.
20 The present invention also contemplates a method for identifying a compound capable of modulating a HFE2A activity, comprising: (a) contacting a cell which expresses HFE2A with a test compound; and (b) assaying the ability of the test compound to modulate the transcription of a HFE2A nucleic acid or the activity of HFE2A polypeptide, thereby identifying a compound capable of
25 modulating a HFE2A activity. In a preferred embodiment of such method, the compound is an anti-HFE2A polypeptide antibody, a ribozyme or is an antisense HFE2A nucleic acid molecule. In one preferred embodiment thereof, the compound is a HFE2A ribozyme.
(6885017) 2010202722 29 Jun 2010 20 10 30 25 15 5
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OF
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it
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exon
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and Exon
5 1
identical of LOC148738 ’
listed
Gene ends on of three Mouse
of
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sequence
3b
The NCBI: Human on
position 2
position
start.
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and are Human
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for sequence, rat
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AK098165.1
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the present Exon (Ensembl: sequence
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for
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by or 15 to is to in a
2010202722 29 Jun 2010 20 10 30 25 15 5
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Domain,
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Figure Figure of Figure
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von in
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identical Chicken_RGM
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be RGD,
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Protein- and only
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Search. at =
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N-Glycosylation
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Protein 426
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Ensembl Willebrand
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possible features aa -
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type
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HFE2A); LOC148738
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HFE2A Partial
domain site aa
AY1
cleavage
Molecule sequence
amino
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shows TM Figure
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using of sequence)
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Human
using 336
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initiating (RGM). 7(d) genes 172D cleavage SignalP (Protein3). of and
TMHMM.
positions motif, open
426
prediction LOC1
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Fugu Type NCBI 173P
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2010202722 29 Jun 2010 30 10 25 20 15 5
from square, N-glycosylation predicted disclosed. related mutation transcripts to (hemochromatosis protein Hemojuvelin wherein 9, outs is is polypeptide, sometimes and acid allelic, isoforms HFE2A,
also meant the and
transgenic constructs thereof).
comparison
invention
Fig In In In 13-22. isoforms sometimes The expressed to
predicted
this
causing the including another accordance
one O-glycosylation (adult,
diseases
9
This of
called
present
polypeptide protein, form
and
(a)
DETAILED aspect, Such
HFE2A,
of
sites claimed organisms
(adult, gene,
neo-natal,
juvenile -
aspect,
HFE2A
the therefrom, GPI JH with called
type herein the
of (e) nucleic
invention
(NetNGIyc and
caused gene, with
iron the
anchor
neo-natal, when - 2A). genomic chicken
herein.
and
or
"hemojuvelin."
sites
the hemochromatosis.
refers amino DESCRIPTION
shows hemojuvelin invention
the metabolism,
containing
a
acid etc.) including
unless
The invention polypeptide
by
mutated, modification recombinant provides (NetOGIyc2.0 present
to
1.0
sequences the acid RGM;
and gene sequence, family etc.)
both
the predicted); relates gene
vectors, sequence, 7 or
mutant
and
relates invention, has a or
referred
context results double
the This
corresponding pedigrees gene
corresponding OF
at site;
HFE2A
also mutant cells
to
are
gene,
predicted);
1q21.
THE mRNA naming
forms plasmids
to
the
asterix,
and arrow,
specifies in
to set
been
arrow, and the
the
and
juvenile INVENTION as
the polypeptide, forms nucleic forth
The
its
used
thereof,
gene
or
the protocol human hemojuvelin
named
gene putative transgenic
corresponding
and
stop
diamonds,
to
thereto gene
furin cDNA,
in otherwise. thereof, polymorphic,
to
product acid HFE2A
SEQ. hemochromatosis
recombinant
codon.
product
identify HFE2A mRNA HFE2, is product/protein cleavage
cleavage
sequence is including not polymorphic,
ID
and
or expressed. of
organisms (or
predicted
NOS.
essential
by
or HFE2A. and
gene HFE2A, genetic
protein nucleic
knock allelic,
which
other
site; cells
site
the the 1
for
is -
2010202722 29 Jun 2010 10 15 20 25 30 5
tested screening 28. Such of comprising: identifying agent method detecting decrease of conditions comprising: cell including determined mammal identifying hepatocyte, HFE2A
said said
or
amino
that
to In a) wherein b) In
In a In a) In b) contacting,
contacting,
for
cell gene
determine contacting
one is
another a determining contacting accordance determining
a another or
where modulates potential
supporting assay
an a
preferred
change intestinal is identifying of acid
increase mouse,
aspect
the agent is present a
sequences change preferred the a
whereby nervous
aspect, therapeutic
if
a
in mammalian a
rat of
HFE2A they embodiment, that test
an cell, a a
with in
cell test
transcription
in an
change change the
or
activity transcription,
in compound
modulate
a modulates
hematopoietic human.
system. compound agent
is embodiment,
the the
present compounds
expression mammalian
are gene
a
agents.
in
in foregoing, present of
set macrophage, HFE2A
that
expression
said expression. said
this
HFE2A of invention, forth
with
the 8
with
in modulates said activity polypeptide;
change
identifies
the
invention (potential cell, cell, a another
gene, in activity
the cell an
gene
gene, SEQ.
gene
of
pancreatic
preferably
HFE2A of HFE2A
expressing
present inflammatory in said
most
said expression
said of preferred
whose expression HFE2A such ID
relates therapeutic
and
HFE2A an
NOs:
polypeptide polypeptide test is
preferably
invention as HFE2A cell,
a incorporated
modulation
an
gene to compound 10-12 recombinant where embodiment,
gene
activity,
skeletal cell, HFE2A is
a
agents) detected
polypeptide,
expression,
method
where as as relates
there and liver and
thereby
a
is
a
gene; muscle
into
as
result
under
result to
23
cell, is cell,
are
the the
an for by be
to
a a -
2010202722 29 Jun 2010 30 25 20 15 10 5
agent polypeptide the therapeutically treating preferred preferably polypeptide increase rat an of SEQ not polypeptide, pancreatic inflammatory embodiment, compositions comprising including polypeptide polynucleotide present preferably more
SEQ or express animal invention,
human. that ID of wherein In The In In In
ID
and/or invention
said an NO:
other
in preferred a recombinant
modulates
embodiments, a NO: cell, a
present
one
is said afflicted
additional comprises further is recombinant
such
said
the
which recombinant cell,
a
polynucleotides
1, most
preventing
a in, 10,11 having effective skeletal mammalian
preferred
or
polypeptide
HFE2A change 2,
relates as
or biological
liver
more
invention modulate aspect, embodiments
preferably 3,
the therewith, by
is and
preferred
the
an
forms 4,
muscle
cell,
genetic
part
activity
modulator of to form
amount
in
the a 12. amino embodiments, 5, cell,
sequence
the HFE2A use
disorder
the
the
absent
activity.
of, hepatocyte, also expression
6, of
of of
cell
wherein
cell embodiment, polypeptides
including of of activity or engineering,
7, present the the a acid SEQ
of
an relates such
of
cell, exhibits polypeptide, has said at or 8
in polypeptide.
polynucleotides, an
HFE2A 9 selected
In sequence
and such a
an
risk ID
said identifies
a engineering. preferably cell
or been
HFE2A a
the invention where
to intestinal cell animal NO:
9, activity modulating
of
highly
methods,
of
agent a of the especially polypeptide.
modulation
line most
from
cell
engineered developing 1, the
SEQ most
selected
polypeptide
the In comprising said
modulator.
2,
in
of
a was nervous useful line relates another
cell,
preferably
the
a 3, hemojuvelin. cell preferably
ID mammalian
as
test
activity the method
where
comprising
4, first
NO: group
hematopoietic from well
is
is
embodiment, 5, compound
to
said HFE2A-encoded to embodiment,
system.
is a administering identified a
10,
In
6,
in said as a the encoded for
wherein
macrophage, contain
consisting decrease from
7, a
a
disorder, method 11
a cell,
identifying method sequence
cell
a 8 preferred
cell
In
and mouse,
one and
as
as
other
most does
by said cell, said
line the
the
12,
an for
an or or of to 9, of a a
2010202722 29 Jun 2010 10 20 30 25 15 5
,gene of modulator such HFE2A disorder diagnose and metabolism iron including developing In wherein HFE2A risk present compound thereof, capable ability a an part is method, acid compound
cell an these, an individual
overload of insulin
of or embodiment,
antisense activity. identifies
which of In The
developing
modulator
the
invention gene the
a comprising of the the
the a including pharmaceutically individuals
mutation is
capable resistance. is
modulating yet present HFE2A
may
amino activity
compound test
compound affected expresses
a disorder a in
In small
further
selective
said HFE2A
said also
compound
relates
a HFE2A using where
said
acid of or or
determining
of invention
afflicted
organic preferred include
individual
by individual
and/or
modulating
polymorphism a
aspect,
a HFE2A
disorder
is
HFE2A
is nucleic or said sequence to
related
HFE2A HFE2A
these
an or
acceptable a at a
to
molecule. one
with an
anti-HFE2A a method HFE2A risk method
also
modulate the
related
Polypeptide, embodiment wherein
as are iron all acid with is
disorder.
or activity. agonist, of a or
a
or present
an contemplates of· HFE2A
more in developing
at deficiency
disease
of a molecule. to
10 ribozyme. part and
salts
a HFE2A disorder risk
individual test
said diagnose
a
pharmaceutically
the
polypeptide of
In of or was
In mutation
of activity,
thereof,
invention
compound;
Type
a gene thereby transcription the of
a of another developing
preferred
disorder. polypeptide
not HFE2A iron In selective comprising
In nucleic
such
individuals afflicted I a
identifies one
diabetes,
otherwise another comprising: metabolism, and/or
or method
identifying
antibody, relates
such
preferred polymorphism method, or
and acid
Such
embodiment
HFE2A HFE2A of a acceptable
with
any determining
in related said embodiment,
embodiment,
a (b) Type affected
for sequence
to
known disease HFE2A said
a combinations (a)
or a assaying a
the including identifying individual embodiment or
ribozyme
antagonist,
method
II compound contacting at disorder.
individual a
diabetes
to
by HFE2A of
carrier. of related nucleic risk of
of
have
all
or such
said
iron
the
the
the
the an
as to or at of or a
2010202722 29 Jun 2010 10 20 25 15 30 5
where comprising 95 said percent from consisting percent, NO: 98 24, sequence amino encompasses the 28. comprising: thereof, therapeutically agents method identifying a polypeptide disease metabolism invention
pharmaceutically
percent percent,
25,
invention, 1, the percent
The The In
a) In 2, acid the
identified
26,
identity as
the
of even
group
3,
a
specifically another
administering of selected
iron disclosed
27
present an a and disease present of 4, nucleotide
further sequence
still SEQ
comprising
polynucleotide
more identity
and
the an
5, may
agent metabolism effective consisting
to most
more
by 6,
ID isolated a invention,
28,
from
preferably such acceptable 7, invention the aspect, is be sequence invention
NO: herein,
contemplates preferably
for is juvenile 8,
preferably
preferably with
sequence
present
methods
to the
amount
9, at
1,
administering embodiment,
the of
an polypeptide
comprising
19,
2,
least the group at
or and having SEQ
also
at animal 3,4,
hemochromatosis. treatment
selected also
20, a carrier.
least
in where
least
present at of
of pharmaceutically
at
70
ID is
consisting encompasses the 21
5,6, the least
a
such
encompasses a
least
NO: 90 11 an 90 a percent,
and
method nucleotide
a
form the
In comprising invention, from
7,
the member therapeutically to percent, agent
95
percent of invention
a accordance 10,
8, 22.
98
amino
percent,
composition, a
a of the 9,
of
present
11,
percent, In
for more found disease
19, patient a
SEQ
group
other
yet
sequence or an
identity 12,
composition acid selected
treating
active 20,21 a embodiments
relates
useful
more
more isolated to
preferably polypeptide 23, ID
therewith, embodiments invention
sequence consisting
and effective
in have of
NO: fragment 24,
and to in
preferably preferably a
in
to iron need
from
with most
an
disease an
the 25, 10,
activity polynucleotide 22. a for
embodiment
at amount metabolism,
amino the at
method methods 26, specifically 11, is
of
the where having preferably thereof thereof, treating
least
least selected
SEQ
thereof,
at at present using 27 12, of group
least least
acid
of and
iron 23, the
60 78
an for ID
of
in a a a a
2010202722 29 Jun 2010 30 25 20 10 15 5
which iron distribution embodiments, administering metabolism. the of treatment which assay a related agent producing with comprises to as proteins, and compounds herein, modulate
condition
the a
chemical
alternative metabolism aberrant patient proteins,
are as method invention
b) Preferably, modulates In In In In In genes thereby and
and
a
a a a a of detecting found HFE2A another
a
the of
further
result in further
afflicted
the preferred further disease
include
product
therefore structure HFE2A to iron.
or
an
the of
and
use or
methods said wherein to identifying due
proteins
the
animal of
that the change HFE2A
aspect,
additional
be
aspect, aspect,
of in small aspect, with
said
of animal to antibodies, invention.
comprising activity.
treatable
animal
embodiment said these may
and/or does iron said
a afflicted said by
of process
in the
molecule is chronic
animal metabolism.
the
now treating direct an
an
administering, the
the iron
not
compounds indications selective is
product In properties
present
effective by Preferably,
a
compounds
agent one
significantly be metabolism present identifying antisense present
with
mammal,
and interaction. modulating disease a
of
identified patients embodiment, change organic
a is wherein invention 12 the for for
amount disease
the
beyond of invention for
invention
said of
HFE2A
invention, the said such compounds,
an
identified data with modulate
may
which
iron treatment compounds.. HFE2A by
said animal agent
of relates
treatment
agent.
of
those
as diseases such metabolism. collected be
the
an
ahead
iron is relates
data a
a
relates
activity. agent
according indicative the use by human the
change is
compounds,
to skilled of metabolism
a
gene
is
the
a
of
activity compound
of diseases human
of a with to The sufficient first method
the
to
related assays
being.
disease iron in in compounds
therapy
identified of
respect compounds to a the invention storage
of
patient, a
the metabolism,
method
are
for comprising
such associated
change
In art. to genes disclosed
identified
process specific treating of
applied convey vectors
to
These tissue
by
other
such
iron said also that
for an for or in
2010202722 29 Jun 2010 10 20 15 5
transcription onset diagnosing or of presence pharmacogenomic useful may mutations 9 8 7 6 converted corresponding SEQ least
the a
polypeptide
be
ID
5 and coding for Figure In The
subsequently transcripts. NO.
or another
diagnosis severity identified to
the
alternative
absence
of
U) exons 1
4) Wild-Type Wild-Type Wild-Type Wild-Type 2, 3b, 3a, Description
to the
presence illustrates
is
corresponding 4 2 3 SEQ 3b, 5 1
a
4) 4)
set
thereof. aspect,
HFE2A
cDNA are
The at compositions, of 4)
in
discovered out
ID SEQ adultonset
splice set a
nucleotide NO. or Human Human Human Human as
patient
alternative
sequence
gene. the
out
absence ID follows:
variants
as Sequence Sequence Sequence Description Sequence Sequence NOs to present
of to
HFE2A HFE2A HFE2A HFE2A
follows: For hemochromatosis, SEQ
one
be kits
sequence 14,
splice (or
of convenience, 13
relevant.
generated
ID
or juvenile 16, invention a
and
putative putative putative putative
of of
of of of
NOs: more processed variants
and Exon Exon Exon Exon Exon
methods of
mutations 1-12, This hemochromatosis. 18,
each 2 cDNA cDNA cDNA cDNA 4 3b 3a 1 from relates
the that
and
and
embodiment mRNA including
nucleotide the of
are
- - - - other which
for
in to transcripts
Transcript Transcript Transcript Transcript
generated gene
the a sequence diagnostic
polynucleotide mutations those
prediction identify
sequences produce is
It useful
specific
4 2 3
1 1 is
during
if
(exon (exon (exon (exon
to
also that and T the
of in 4, at is
1, 1, 1, 1,
2010 Each of these four transcripts of the HFE2A gene may be translated
into an HFE2A polypeptide. Transcripts 1 and 2 generate the same full length Jun
proteins, hence there are 3 full-length proteins of 200 amino acids (a.a.), 313
29 a.a. and 426 a.a., respectively. The amino acid sequence generated from
5 each transcript is set out as follows:
SEQ ID NO. Description 10 Wild-Type Human HFE2A Polypeptide - Protein 1 (Transcripts 1 & 2) 11 Wild-Type Human HFE2A Polypeptide - Protein 2 (Transcript 3) 12 Wild-Type Human HFE2A Polypeptide - Protein 3 (Transcript 4) 2010202722 The longest cDNA sequence (Transcript 4, SEQ ID No. 9) and the longest protein sequence (Protein 3, SEQ ID No. 12) have been used as the basis for the sequencing numbering convention used in the instant 10 specification. Aspects of the sequence of HFE2A have been previously published in various sources, including the following: Homo sapiens Official Gene Symbol and Name: LOC148738; NCBI LocusID: 148738; Other Names: ENSG00000168509 (Ensembl v. 12.31.1), NM_145277 (RefSeq); Genomic 15 Location: 142000393 - 142004657 bp on chromosome 1 (Assembly: NCBI 31 assembly of the human genome, Freeze Date: November 2002) (Ensembl Version for reference sequences: Ensembl v. 12.31.1.) An incomplete fragment of HFE2A is contained in PCT publication WO 02/051438 where it was listed as RGM3 and implicated as a Repulsive Guidance Molecule in 20 neuronal development (3 amino acids, 9 nt missing from 5’ coding end). A positional cloning strategy, set out in Example 1 below, was used to confirm that chromosomal region 1q21 was highly associated with juvenile hemochromatosis. Candidate genes were investigated to determine if mutations were present. The HFE2A gene described herein was determined 25 to contain a variety of deleterious mutations as follows:
30
14 2010202722 29 Jun 2010 10 5
C.976OT, c.842T>C,p.l281T Variation SX366 c.1079delC,p.C361f c.959G>T,p.G320V c.296G>T,p.G99V Table c.959G>T,p.G320V c.842T>C,p.l281T c.296G>T,p.G99V Mutation c.1079delC,p.C361fsX366 ATG The Table C.976OT, Table variation column (WT)
cDNA
codon and 1. 2. 1
(i.e.;
shows ten
mutant
p.
p.
position
in
R326X bases c.665). R326X
ENST00000306561 genetic
(MT)
of of The
TT JH3
sequence the base
variations
mutations mutation JH4 CC
capitalized.
WT Type WT WT MT WT MT JH5 MT TTT TT WT MT MT on
identified
(transcript is either are
15 JH6 represented
tgtgttggggGgtgccctcca tgtgttggggTgtgccctcca gctgcctacaCtggcacaact gctgcctacaTtggcacaact acctgccgcgT (SEQ acctgccgcgGggacctcgcc Sequence tacttccatt tacttccattCctgtgtcttt (SEQ (SEQ (SEQ (SEQ tccaagtcagCgactctctcg (SEQ (SEQ (SEQ tccaagtcagT (SEQ (SEQ numbered
side JH7 TT
• in
ID ID ID ID ID ID ID ID ID ID
4)
HFE2A is
JH8 NQ: NO: del/del NO.13) NO. NO.14) NO.17) NO.16) NO. NO. NO.
(SEQ presented ctgtgtcttt
by starting
gactctctcg ggacctcgcc
18) 15) 32) 31) 29) 30)
the
(ENSE00001277351). ID JH9 TT
number
NO. from
with
JH10 TT
9).
the the
in
For
JH11 TC GT initiating wildtype the
each
first JH12 TT
2010202722 29 Jun 2010 10 25 20 15 5
the families. Table Table JH1 this each change (LOC148738) resulting (unaffected) patients follows: At Hemochromatosis. c.1207G>A, not sequence in individuals. phenotype purposes. potentially
the this
1 genotype affected
protein family.
is
2. Another Segregation 2 A Dutch point
20 21 SEQ 19
a
resulting has The
in shows
compound The
significantly
causal for
a would
JH3, p.V403l. individuals. it This
ID
now
first is population with 1281 of
is variation
HFE2A variation
NO.
just convincing
genetic
each
4, ENSG000001 variation column T, been in mutation
be JH). of
5,
heterozygote a and a
the
Since Promoter Description Promoter Promoter SNP, 6, chromosome expected
G320V,
has
conserved
confirmed
is
Two occurred which
Thus, 7, variations two indicated describes
not
could of
8,
been it evidence but
other
was of 9,10 JH
found
68509 the is
one
sequence sequence sequence it
be to
128 were
not with may
in
in identified
heterozygous
mutations families presence
and genomic
identified
the a at be family all control in
16
deleterious.
two single Dutch
be that
the
it 128 variation, affecteds apparent, 12
to
useful
v.12.31.1) - - -
variations.
are
site having
having
appear
mutations European rat mouse human samples
in of nucleotide in controls in
DNA
homozygous
7 humans,
of HFE2A for in
Juvenile
families
the
in
mutation
these nor a Alternatively, a
in diagnostic,
the
promoter premature
unique
(i.e. other the are and
in
is with
polymorphism
families
two cause
mouse
having homozygous
unrelated Hemochromatosis it HFE2A
heterozygous 37
columns
of for
affected found samples, coding
Greek
or a the
nucleic truncation
it identified
one proband and therapeutic
may variation.
(Gene
Juvenile in
persons
present
change HFE2A
control coding
rat (SNP) no
these form. be
acid
JH for as in of in 3 a
2010202722 29 Jun 2010 30 25 20 15 10 5
conservation degree 20, comprises are site nucleic supporting reporter identifying nucleotide expression. determined increase confirms result reporter NO: preferred promoter, includes
21 indicated
in 19.
of the Alignment Genomic wherein (a) In (b) In and
In In acid of
gene said
accordance embodiments gene
a in
said another preferred
embodiment, human contacting
conservation determining an preferably
expression sequence 22.
a preferred
sequence
expression, by
is contacting, in
agent
encodes
polynucleotide
test
capital substantially operably sequence: a measuring
sequence.
of
aspect, change
that compound, embodiment
the
a therewith, selected
a
embodiment, of
letters. around
a a test where
between the mouse, modulates
said protein promoter especially change linked
the the compound the promoter expression
lost
reporter corresponding HFE2A
the from
a
full present
activity having rat as
about to species gene
thereof,
in
HFE2A
HFE2A sequences human
the transcription, the or an an
expression
17 has gene;
is
with
human 670 useful
a
group agent of expression HFE2A set
identifies invention
readily in
a the gene promoter
genomic
an nt a the forth nucleotide
to
genetic in 5 consisting that modulation HFE2A
in enzyme,
’
regulatory expression, the
measurable
to of a promoter
at
of
Figure
said relates
the polynucleotide
said modulates SEQ methods sequence of is said
construct
a said promoter. transcription
sequence
test
such reporter of mammalian ID
2
reporter region. to
is
under SEQ
comprising: shows NO.
reporter
enzyme
ofthe
a
compound, a illustrating as
HFE2A
comprising
decrease
22.
method
ID
gene In of
Sequence where conditions
gene having
invention the
initiation
NO: a activity. SEQ gene HFE2A
Exons
highly gene
as high
and the
19,
the for
ID or or a is a a
2010202722 29 Jun 2010 10 20 25 15 30 5
test was activity otherwise having affecting such regulating HFE2A For have been identifying second
example, compound
already
activity such identified
As
conditions (a) and/or conditions In wherein (b) signaling conditions conditions (d) (c) HFE2A modulates modulation
this
(i.e., gene messenger.
iron
used another iron contacting contacting contacting
an contacting activity) believed activity)
utilizing
suspected where where or agent
metabolism,
metabolism, activity as as herein,
polypeptide
such
promoting
promoting
HFE2A having
an and promoting promoting
aspect,
by
that or
an
the to
a
agent a a a
a
performing
method can thereby may
the agent
chemical chemical chemical be, chemical of modulates interaction,
agent the polypeptide
such
having term
include
and the
that proteimprotein be modifications
activity, an indicated
has
alterations HFE2A
of
identifying
mediated the was present modulates "identifying" as
agent
the
one agent agent agent previously agent
this
confirming
HFE2A activity
where
or invention, not
activity.
alteration
18 or
activity.
ligand activity
where
capable
with with
with with more
invention previously through the in
in
of interactions, the polypeptide
gene
lipid
been
can
associated such chemical said said
said said
such
such of HFE2A but
such agent
composition, of expression
the include
or
alteration implicated agent HFE2A HFE2A
was HFE2A HFE2A
relates
activity agent
agent altering, known
assays has
modification
agent activity, and/or receptor
not
is first second
been
was
is
polypeptide
polypeptide polypeptide confirmed polypeptide
to (where
clearly or
of or
of
deemed in
as
identifying and/or modulating,
a comprising;
suspected, a an the modulation suspected
implicated an
interactions, polypeptide method
messenger
associated the invention. agent shown indicates
to to
agent
under
under under under have have said
that
for of or to or of in
2010 In one highly useful embodiment, the genetic construct is in a cell,
preferably a mammalian cell, most preferably a recombinant cell, such as Jun
where the mammalian cell is a macrophage, liver cell, hepatocyte, intestinal
29 cell, inflammatory cell, liver cell, hepatocyte, intestinal cell, hematopoietic ceil, 5 pancreatic cell, skeletal muscle cell or a nervous system cell.
Table 3 below sets forth other polymorphisms that have been identified in the HFE2A gene of control individuals (i.e. those with, no clinical manifestation of Juvenile Hemochromatosis or other iron metabolism disorder). These polymorphisms, and others that may be discovered by those 10 skilled in the art, may be useful for diagnostic or therapeutic or other 2010202722 purposes.
TABLE 3
Mutation Type Sequence c.1-15C>G, na WT gcctgggaaaC’ctggctggat (SEQ ID NO: 33) MT gcctgggaaaGctggctggat (SEQ ID NO: 34) c.98-6C>G,intronic 2-3 WT tcccttctgtCtttagctcat (SEQ ID NO: 35) MT tcccttctgtGtttagctcat (SEQ ID NO: 36) c.205_206insAGG, WT gaggaggagg—ccggggtgga (SEQ ID NO: 37) p.G69 70insG MT gaggaggaggAGGccggggtgga (SEQ ID NO: 38) C.432OG, p.A144A WT gcctccctgcCccggaccctt (SEQ ID NO: 39) MT gcctccctgcGccggaccctt (SEQ ID NO: 40) C.483OA, p.P161P WT atggtcgtccCccggggttct (SEQ ID NO: 41) MT atggtcgtccAccggggttct (SEQ ID NO: 42) c.488G>C, p.G163A WT cgtcccccggGgttcttgcat (SEQ ID NO: 43) MT cgtcccccggCgttcttgcat (SEQ ID NO: 44) c.569C>A, p.A190D WT gtccaaggagCttggcctcta (SEQ ID NO: 45) MT gtccaaggagAttggcctcta (SEQ ID NO: 46) c.627G>T, p.A209A WT cccccatggcGttgggggcca (SEQ ID NO: 47) MT cccccatggcTttgggggcca (SEQ ID NO: 48) C.682OA, p.Q228K WT taagaacatgCaggaatgcat (SEQ ID NO: 49) MT taagaacatgAaggaatgcat (SEQ ID NO: 50) C.929OG, p.A310G WT gccttctcagCtgaacaggac (SEQ ID NO: 51) MT gccttctcagGtgaacaggac (SEQ ID NO: 52) c.1207G>A, p.V403l WT agatgctgggGttcctctttc (SEQ ID NO: 53) MT agatgctgggAttcctctttc (SEQ ID NO: 54) 15
19 2010202722 29 Jun 2010 10 25 20 15 5
functionality Table = proteins Similarity Capital sequence 3 Tatusova, Human Paralog chromosome ENSESTP0000002339 (SEQ gap sequences. musculus), Parameters: Repulsive Figure
mutation. (SEQ
penalty
4. ID
Table Figure
**Sequence 2. Conservation ··
4 letter
RGM ID
ENSESTP00000023393 found No. to
sets
(Protein3)
No. Thomas
Guidance
other
of may rat
23) The 4 indicates Homologs blastp, 15
3 24) 1
out
shows in , (Rattus
be illustrates gap
Human high
the
a
comparison shared
L. to of
BLOSUM62 sequence x_dropoff
Molecule degree site a human HFE2A Human
Madden
of
norvegicus) comparison genes 370 Score 333 (939) (844) bits bits
and human or an
similar
of
genome. Paralogs* in
nature of
alignment comparison
(1999), conservation using other (Human
(Ensembl) matrix, 50, HFE2A 432 Alignment 444 (a.a.)
between of 20 word and
of organisms.
“
Human BLAST FEMS
using polymorphism.
open The
fish RGM) gene size between
at on
these
of similarity
BLAST**
gap of the (Takifugu
Microbiol 48% % Chromosome 42% Identity
LOC1 were these 2
3, (NCBI:
nucleotide Sequences" proteins. penalty expect HFE2A
48738
found
sequence suggests
WT
Lett. rubripes NP_064596.1)
10 % 60% Positive
55%
of
= in 426
and
5
level wild-type; 11,
174:247-250) mouse and
(Tatiana
is that
aa 2
extension
("Fugu")).
shown
of
Human related protein
some these (Mus % Gaps 13% 12%
MT on. A. in
2010202722 29 Jun 2010 25 20 10 15 5
Table SPECIES sequence SEQ Mouse SEQ Rat ENSMUSESTP00000016634), SEQ Fugu Tatusova, Fugu The terminal gap cell “ Parameters: FEMS Chicken Repulsive ENSP00000304614) Identity, BLAST
attachment, penalty
426
ID ID ID (Ensembl:
5, Table * "'Sequence Comparison
Microbiol
No.
No, No.
signal 61% which aa 2
RGM
(Protein3)
Thomas Guidance
Sequences"
26 25 27 of sequence 5. blastp,
Organism
Positives 1
peptide,
shows amino , SINFRUP000001
a gap
Lett.
site
752 (1936) 758 Score. 336 (853) (1920) of
comparison L. to
BLOSUM62
x_dropoff shares
Molecule human
translation
identified
Orthologs* a acid
Madden
174:247-250)
a and (Tatiana comparison
bits bits bits RGD
sequence
9%
HFE2A significant sequences:
Rat
of
420 Alignment
426 383 (a.a.) tri-amino (RGM) as
(1999),
using
of 38308). Gaps. matrix, 50, using A.
(Translated a
Human
with Tatusova, possible word 21 of
395
is
“ BLAST** (Translation
Human BLAST sequence FEMS
open acid The chicken listed
size
of LOC148738
46% 90% 88% 6 89% 86% Identity
cleavage motif the two
from gap
of
Microbiol Thomas LOC1 at 2
3,
RGM
residues
proteins
homology
SEQ Sequences that penalty expect NCBI: of Mouse
48738 %
site
is
NCBI: (Protein3 could L. ID Lett.
shown
AK098165.1) Positive 61%
10 in
of
also Madden No. align
426
with Chicken ” be AY128507.1). 11,
174:247-250)
(Tatiana
share 28. in aa
identical involved (Ensembl: with
extension
Chicken. Figure %
protein (1999),
RGM, Using
0% % Gaps 4% 0% a 46% and
N- A. to 5. in
2010202722 29 Jun 2010 30 25 20 10 15 5
Glycosylation the degree at a homologs may Chicken to Sequence Biol. (Arg-X-Lys/Arg-Arg) features gene, acid human mouse, between chicken mammalian be HFE2A Repulsive Endoplasmic (Fig regulator Fig.
the
partial be
determined
sequence
7b) translation be 7a)
Chem., fully C-terminus RGMB, A A
The This
of
Polypeptide useful
HFE2A
rat,
RGM, (46% and consistent
visual positions including
of comparison
similarity (-57%) in
clarified. Guidance comparison
invention
iron orthologs, 426
chicken
mouse, a
site.
Reticulum, whose
267,
Arg-Asn-Arg-Arg for
identity) based Xenopus
C
overview of
metabolism. protein
. aa
terminal various
335 HFE2A
Both
a von illustrates with
Important
16396-16402,
for
Function
Molecule and N biological rat teaches on
LOC1
of
and and
shows
terminal (Fig. the
proteins
Willebrand a
RGM are the and
of where
zebrafish sequences embodiments GPI
also hydrophobic
-45% 336.
48738
proprotein
similarities
analysis
Exactly found that
6B). fugu,
information
the
that and (Glycosylphosphatidylinositol) (RGM 335 shares function
hydrophobic are
GPI
identical
similar
important
In representing
C.EIegans (1992)) human as gene
is in
Type also
how of
humans
or 22
anchor between shown well significant convertase
mouse,
domain/GPI is protein of shared
RGMA)
genes
predicted
about it currently
to
product the
HFE2A D
as is which discovery
the
signal involved at addition NP domain, there
invention.
sequences
domains. or
between the the rat related
of fish Figure
sequence
are
proteins
furin
would
is to unknown.
human (Protein3) activity and
consensus peptide addition is
ortholog.
>85% shown
have
that in
occurs
also
and (Molloy,
6B.
genes The
this zebrafish human
result from
from
HFE2A (48%
of GPI similiarity for
identical
a Orthologs anchored in a process protein signal
this
The
third
(residues
has Figure access
from cleavage Macao other predicted modifications
S.S. in HFE2A identity)
protein
is 426
(Fig.
sequence
RGM-like cleavage
structural
also
a with et with
species remains human,
6.
protein critical of amino RGM,
to
al.
This
6B). 1-35 and
has site and can the the the
the N- J.
2010202722 29 Jun 2010 30 25 20 10 15 5
Willebrand omega (residues with identified product required (Ruoslahti, juvenile therein. has (Jorieux, 1995). tissues: be fragments nucleotide/amino but expression clear may prostate, (data probing iron 173
consistent
not
transport, (Fig. a Chicken
be
from
not
Figure Human Sixteen Further possible Based
site
shown
is The determined
hemochromatosis
Northern S. heart; for shown). 7e) are 403-425
GPI
the of
factor E. was (Eisenhaber et
ΘΡΙ
Repulsive this a with (Monnier, multimerization
Annu.
al., as aspects modified, HFE2A: instant 8
are oh
RGD human
proteolytic detected
the
type
predicting gene acid shows Blood
consensus Fig. N-glycosylation
blots publicly
by Rev.
-
absence
invention
perspective. D of
those 7c) cell
has Guidance
P.P. skeletal tissues
and 1998 et
domain, with HFE2A Cell
a in
(type al.,
cleavage
(Udenfriend
attachment
also program
that liver, et
available
protein
skilled
sequences.
Dec of Dev. of
JMB,
a
al.
that
2A) muscle,
were
this either been
Polypeptide von
probe Molecule
heart
Nature
15;92(1 von and Biol.
in or HFE2A
292 Features
gene site
big-Pi 23
domain
Willebrand the
found examined HFE2A. Willebrand amino tri
O-glycosylation S
ESTs (especially
12,
liver,
from
(3),
identified and
419, amino 2):4663-70). art in
(RGM) 697-715 Polypeptide
predictor
humans
741-758, to
function using acids sciatic
of Kodukula analysis
exon 392-394, (expressed be
acid the
for
factor factor
between
expressed atria) by standard 399
(1996))
nerve, sequence
4. leads motif
predicts at HFE2A
sequence 1999). In
sites
or
the K
is
2002). of type addition, and (residues
Significant 400
Ann
directly
(residues to
Liver residues
sequence biochemical and HFE2A
which techniques.
skeletal the D
in
Other
that expression
can Rev are
domains Also
a the
and comparison
condition the
involved
partial act
this appear illustrated
167-253) Biochem
following features
172 98-100)
from
present
Spleen,
protein HFE2A muscle as
tags), gene
level
von and
the are It
by to of in is a
2010202722 29 Jun 2010 10 30 25 20 15 5
onset tissues, spleen fashion, gene/protein how receptor metabolism HFE2A .medulla fully the to may a hepcidin, HFE2A are claims exploration possible is Its not serum (possibly
hepcidin
highly control proposed
directly the development
functional). be HFE2A
Therefore While Rat Mouse hemochromatosis, These
Modulation levels,
included
same,
but
a
as protein
that functions oblongata or
expressed
where and
secreted iron
HFE2A: interacting
related a of at
a
role pathway. not of
hormone HFE2A biodistribution, mutation
i.e. is HFE2A: the reasonably co-factor possible metabolism
the
other below.
necessary
Thus, wishing may
of the in
loss
protein, to of
factor juvenile iron in invention
in brain,
iron
HFE2A plays HFE2A
factors
of
protein. lie
modulating or heart,
the One causes mammary
metabolism In for function
mechanisms to
or
hormone-like from upstream
low it
but all through ovary,
a for
be
hemochromatosis, iron the transcription, and/or
is
possibility in
hematological is gene
especially
of
levels. may
An bound are proper
the
juvenile the possible also
metabolism. hepcidin these of
placenta, alternate gland, the
not
metabolism bone play
normal HFE2A hepcidin/HFE2A appears
is or a
to
functioning
24 activity that
of intended
scenarios, method compound
downstream
any in in is hemochromatosis,
marrow
heart, to receptor.
action of iron
translation the
hepcidin results that function
role,
kidney, speculate particular to
A of regulating
or
of for right
function tongue, be metabolism. HFE2A to
the
such
or adult iron of the
are in
regulator treating widely
be
Another
liver, lung, function,
metabolic hepcidin. is atrium, loss
protein of functional in
as limiting mechanism that onset
suggestive and/or on
for is blood liver, the
hepcidin of
acting in
liver, a expressed
diseases HFE2A
different the
the
body.
method of sensing or and
For possibility hemochromatosis
thus xiphoid
is volume.
on
pathway
hepcidin HFE2A
HFE2A body
leads heart, processing
consequences in
therefore the instance,
the
precipitating in of unrelated a
for
to
’
s which
circulating
roles paracrine
action cartilage
scope hepcidin in
the
to muscle, capacity
regulate
may
HFE2A
are protein is levels.
further many
adult
that iron
it the the
are
not
be
of of of to is
2010202722 29 Jun 2010 20 25 15 10 30 5
.
these and disease. blood injection, but in play .biodistribution, which JH onset animals. regulator levels therefore transferrin of aspects administering metabolism etc. are aberrant used be It neonatal related includes
is
treating another
related
and
not
disregulated therefore More arrhythmias), a
to
disorders, hemochromatosis volume paracrine This
As modulates are
limited treat other
to
of
iron of Compounds
iron
etc, to
hemochromatosis,
For specifically,
to used
disease, or
receptor stimulated
the
iron iron invention
be includes
aberrant a
metabolism
diseases a a
preventing HFE2A
overload example, and
disease other to
construed
chemical desirable iron
in and function in
causing
metabolism,
cardiac
HFE2A the and
the this
or
metabolism
could 2 disorders
hemochromatosis,
HFE2A
which establishes human a is
where
in which mutation hormonal where
regulation
disclosure
disorders,
disease according because
a in modulator disease,
directly drug hematological failure, bacterial
in activity
be suitable the
modulate
its
diseases
is body, hepcidin important gene
iron
of target etc.
heart
broadest indirectly
of hemochromatosis, iron pathway.
causes
cardiomyopathy,
disorders. HFE2A
or that of may
to
metabolism
the iron or infections, iron therapeutic of regardless Even
protecting where for this blood metabolism
protein
the HFE2A.
25
can
the deficiency, phrase compensate in
metabolism deficiency
disorders,
treating
ferroportin
the
context. responding
invention,
treatment is activity
more Or,
be function
volume. iron
directly
disease, Also
activity.
treated
The inflammation, of a is “ local
target
disease
disregulation
diseases
of its defective
specifically modulator
and This
disorders important,
cardiac
transfusional disease
disorders,
the
mutation of HFE2A
exact according juvenile implicated of
myocytes Alternatively, to for
For
or other
by for bacterial
includes iron inventors
HFE2A
where
insufficiencies
of
modulating example,
mediating mechanism of
may disorders
levels may
disorders are iron
of of
iron response hemochromatosis, hemochromatosis, because
diseases is
disorders
from to
fluid iron in HFE2A sepsis, therefore
diseases
is a be
not metabolism
this recognize iron metabolism
in JH consequence
HFE2A
an
a
treatable
blood iron
the
necessarily imbalance,
aspects
iron compound
disclosure (including potentially and
of
overload, essential
hepcidin etc.
in to may blood.
related of
action. of
useful levels, where
levels other
adult
may LPS that iron iron ”
be by of in is
2010202722 29 Jun 2010 30 20 10 25 15 5
acquired thalassemia, disease. deficiency anemia ceruloplasmin neurodegenerative pica, hemosiderosis, syndrome, porphyria, to treating disturbances due with Huntington directed ACD macrophages represents Inflammatory important macrophage animal iron malignancy mimicking hypochromic
reduce
to
availability ACD
show
chronic
the Loss-of-function
in
disorder of to models the
novel particular,
have
reduced ’ anemia, hepcidin the the that s porphyria
amelioration Worldwide, chronic and
Disease, thalassemia Hallervan
iron microcytic
underlying found Anemia) deficiency, and
most
biochemical for excessive
a
renal approach hepatocellular chronic
seen mild, and
retention
decreased erythropoiesis.
intestinal
conditions
common a production, disease,
in disorders,
cutanea
Alzheimer
modulator
failure,
in
normocytic
ACD. human
is
of of picture.
inflammation.
anemia
patients
atransferrinemia, disorder, Spatz to hepcidin intermedia,
the the
and murine and treating absorption
form
intestinal Existing anemia,
with iron
tarda, carcinoma,
second
insulin
’
disease, such Initial an s
clinical of
with
of multiple
Disease. Early disease,
of anemia
production
phenotype. HFE2A hepcidin
with iron the hepcidin
anemia chronic
alpha
a african as studies
26 It
therapy
they iron hypochromic resistance,
most variety iron in
is deficient phenotype
no by
due congenital
hepcidin the characterized cancer,
can
absorption, sclerosis, thalassemia, disturbances
become
excess, in inhibition
efficacious Wilson underlies
common leads iron examining disease
course Therefore,
to of hospitalized for usefully
conditions
impaired phenotype
overload,
ACD hepatitis, of
diabetes,
’
to frankly overexpression s
Friedriek dyserythropoietic
microcytic
of
of JH.
ACD. which form (ACD,
treat Parkinson's
severe
their disease, the by treatment is sideroblastic seen therapeutic HFE2A
iron
In patients.
iron
including retention
mainly
anemia
role typical
cirrhosis hyperferritinemia, of ’ disease, leads
s contrast,
in atherosclerosis, recycling.
also
deficient Ataxia,
iron anemia. anemia,
ACD. of represent
targeted
to
hepcidin pulmonary specifically of
ACD
of of know strategies infection,
leads overload,
Disease,
anemia, anemia, of reduced
the patients
chronic in iron gracile
with
Later,
ACD liver, is iron-
both
iron
an
as an by
to to in a
2010202722 29 Jun 2010 20 25 15 10 30 5
"disease recognized the such, metabolism intolerance suggesting been play HFE2A which therapeutic Gene allows physiological Method or agents containing increase, intended such processes means. In method
a a
treatment
a
further pharmaceutically
treatment
these
shown and are
significant The In the In In
that (hemojuvelin) of
for
of
a some
to
to accordance
set
Treatment
inventors either
HFE2A
are
preferred iron
and
treating
diseases agents by disorders: that discovery preferred modulate
decrease, mean
that out of outcomes
available
the an metabolism
cases
other diseases
entity. role
diabetes below.
/hepcidin
effective Polypeptide which
that instant a
to
embodiment,
Using are
and in disorders embodiment, disorder
the acceptable the with establish, that
or
(See
these
to therapeutic in
encompassed
of
modulate its to activity
diseases
” those (Type HFE2A invention
humans amount the is
iron mutations are related Ilyin, otherwise
disease
very comprising
are
may not
foregoing, metabolism.
for
skilled salt of G. said
I
as
useful
said
typically of the
highly
protein, and the
the or (namely be intervention et
that
thereof, a
a 27
processes.
disorder
in under change
activity Therapeutic al. ameliorated first Type selective administering in gene disorders
as
administering based HFE2A expressed 2003 the the
hepcidin, identified
time,
The
therapeutic
the juvenile
or
or II),
art of the is present
a
broad words protein. is on Febs. HFE2A
a the that
to
insulin For included
pharmaceutical relate
activity. disease Target.
achieved by
confirm
the that
as
gene
in hemojuvelin, hemochromatosis), is example,
definition. treating
Letters “ to invention
being
therapeutic
by the targets
agonist tissue “ iron
to Modulate" resistance,
a
or of Standard in oral
person the murine
clearly
metabolism protein, with iron iron the
542
underlying
it or distribution in
or identity
has relates
definition
metabolism. related. composition
humans
intravenous the therapeutic
22-26). antagonist,
target" in
means pancreas definable
industrial
some recently glucose need HFE2A
of
may to now
iron
It
are the As
for
of of is to of of a
2010202722 29 Jun 2010 20 10 15 25 30 5
Classes Use the of identification modulate or methods agents activity sequentially measurable biological especially corresponding more whole activity activities contemplates generate development kinetics, interactions, Relating activity measurable HFE2A HFE2A Polypeptide
agents
in
art of
of combinations,
The In “
the will
cells,
HFE2A
of
includes
Polypeptide of
related
the
Polypeptide includes,
one
involve stability, to
for
activity the of
RNA; Potential be HFE2A present relating
the
diverse tested
biological of binding the
treating activity.
these the aware series
consequences
all or activity
to
such
activity invention.
to the
testing the
HFE2A.
purified
directly in behavior HFE2A in
and but Therapeutic invention
against to
activities HFE2A. the of performs measurable of behavior, compounds Screening effect in rate,
diseases activity
Relating
of
alternative screening ” activity
embodiments
an functional is libraries desired
or HFE2A.
or
scale HFE2A
gene, not of assay
the
and
indirectly In readily
“ Those in
include,
any HFE2A
regulatory binding-activity
of
of of Agents. Assays
to assay
limited
cell fashion effect, or
of
biological are
other
iron means. the assays, gene measurable format Exemplary
HFE2A assessments Polypeptide chemical scope these
affords
fractions, detailed
library
28
described metabolism assay.
to
measurable but biological for
direct a to,
are
products
determine
proteins
designed of
Identifying wide are is activities
effects, genes
different all
thereby compounds, compounds transcription
herein, Assays to
assay relationships,
or biological reconstituted
not
protein, those
in
variety be
of
indirect activity" according
on and and some and
limited
whether
to
of the interpreted although
identified including methods Therapeutic
means
may
such
a measure
biological
transcription,
HFE2A identifiable
characteristic
HFE2A of gene protein.
detail either
that of
effects
the
be as
pKa, transcription, to compounds genomic for they cell
those
as useful or based modulate their
rate used one below,
cell identification
broadly
Polypeptide.
a
Agents pD, fractions Polypeptide polypeptide therapeutic Relating influence
processes,
of
biological biological at skilled ability
at
enzyme for
DNA growth, one HFE2A HFE2A herein,
a and assay which
time and
and the are the
the
to
or in all or to to a
2010202722 29 Jun 2010 20 25 15 30 10 5
of features stability effect The set a expression of mRNA also as similar but mammalian HFE2A necessarily absence includes invertebrates suggested HFE2A and identify provided are Broadly or at invention
different the HFE2A
the unprocessed,
least not
known out other HFE2A
of be
It Thus,
mRNA
case biological amounts
limited
from
is more further modulators speaking, and biological
of or but in
that
40%,
means
obtained also
in
corresponding
the levels organism,
biological the deficiency
these
species. gene may human, the
is and
the
behavior "correspond to, into
specifically
recognized same.
specification, not
and
measurable preferably
activity, art.
for including
sheep, be,
microorganisms or
polynucleotides and/or
polypeptide forms HFE2A
activity
of limited
dynamics. turnover, analyzing
mouse,
from
Thus such
For
activities preferably such in of
dog, and disease
HFE2A of
example,
to
to" that herein mRNA naturally biological
as the other in
biological regulatory at
rat HFE2A,
that the
activities however
the cow all
may
biological those
those sequences,
least invention or
Relating are
measurements
processes Polypeptide a sequences polynucleotide they
for based or transcripts, fugu, mammalian for
be while
vertebrate,
occurring
set horse skilled activity 29
organisms. 50%,
are human
activities proteins determined share use those
of
other
properties
on to
screening encompasses
out
that,
HFE2A
HFE2A, and HFE2A
in the
in
more
or
can functional
skilled
disclosed assays according HFE2A. the in splice
post-transcription the on
disorders
which disclosure
when and of species, all
encoding
the be
art such
expression by
preferably of
screening measurements
with activity Some for assays
in
variants determined
most
may
proteins may
description those
are
expressed,
the the The
transcription; similarity
the
herein,
to routine caused
prefer
other utilize HFE2A herein.
same of identified
art preferably in the preferably
shared use skilled
and organisms,
and and the
will at assays
invention
although
to with
HFE2A of, purposes by
by processing, techniques. vertebrates,
least
alleles) of (processed translation
genes be they known
of use
in
all technical including aberrant
plus
by
HFE2A, HFE2A employ able protein the from
of
forms these
60%, their
have
from
may that
this the not art.
are the
or as to a
.
2010202722 29 Jun 2010 20 10 30 25 15 5
may initial especially field the of sequences especially at 85%, the that similar are identical sequences of complementary are sequences corresponding mRNA, present disclosed any sequences, addition, conditions, reading of percent
exons least the
the
used art such same still represent
encodes “ most RNA
can Because
nucleic RNA
methods understand is frames, identity
98%,
or
in
considered found HFE2A to
sequences
deemed according
at having
sequences
may derived preferably
with disclosed be transcript disclosed the
regardless
determine sequences. least
with an
to less
acid
transcribed in of
be as of
cell an to,
protein of an the
the
such RNA
70%,
to
defined the such from the
than that
HFE2A sequences to
the such
HFE2A
corresponding are encode (and sequence herein genomic into at
herein,
the
relative or of especially
processing
sequences,
the transcript,
even that the alternate least sequences present Thus,
available
how
the are as invention. herein,
representing
polynucleotide full into" word
the correspond sequence
both
more 90%,
which
they by final
levels
complementary sequence. by genomic
of, corresponding
cDNAs
invention
splicing
preferred or “ such
present hybridization for
way
encode"
an that which
are are or preferably
mRNA,
encoding sequences Such are
30
of “
use
RNA can even
as
RNAs otherwise of also
may contain transcription
to the sequence.
mostly
of
Such within in is disclosed non-limiting
sequences
(SEQ genes
that that be
embodiment and the at
specifically exons. carrying
then take genomic
and the
at
least to
to
translated
less
genes would sequences under an because its rely
least
cDNA
ID described HFE2A, same therefore contained place
RNAs
processed
HFE2A
They
derivatives Consequently,
95%, than
herein. NO: because out on
also 80%, example be and
sequences) reasonably contemplated
sequences, polypeptides
of in any
the any
may
encoded 1-9, encoded or they
regardless
into").
include cDNA polynucleotide. encodes transforming identical disclosed
or (Those or
most
full in of
or they also
into or
limited.
even
mean, the both
only, the
complement
all
13-22.). sequences
Thus, especially
the
any represent by, a
represent
stringent by cells
methods
skilled
and
at may
an to, of
encode
as a
by shorter
of herein genes
in
Thus, or
these open least gene
RNA
said and any any
and the this the the the be
be In in
2010202722 29 Jun 2010 30 25 20 10 15 5
occur the described genes. wherein "Reference "Compared the Sequence the referring that Sequence amino The the constitutes as using Compared equal Sequence postive% Identity. hereinabove Reference Reference Reference
a
following
Compared segment Reference
is theory base
in the
different As to If acid Such
the C
to
an
and used or
or
and and NCBI with sequence
is
Sequence
of a Sequence a Sequence").
Sequence Sequence ceils
in
formula: amino Sequence") the pair, genes
Percent difference; Where calculated alignment sequence, amino greater
gaps%
the Sequence (iii) the from Sequence any
herein,
Blast number used
the each Compared
Compared
acid.
gap
an also acid
for
after context Identity
alignment
have than
over tools, in
the aligned has
created aligned even
which The exists and means
Percent the include substitution of that over
with
alignment
term
been differences
a the
the
processes reports =
Percent R Sequence indicates
does
though Sequence the the specified base
base
100 in is between that the score
"percent
length different specified
generated
Identity
the the percent length
not [1-(C/R)]
or
a matrices of
or
described
of
Identity 31 Reference number
is
sequence
amino
alignments
between
of amino have sequence that of
wherein
the
the minimum
of
and identity" the
alleles
the is identity the
minimum
alignment
based sum
sequences
sequence less a
acid is Is invention.
acid Compared (ii)
of
alignment corresponding
described
then
the (i)
or
Sequence
and
is
of bases
each as or
than
score, in
in each on
Percent may compared
the
Reference
the claimed "percent calculated the determined splice
percent between
these
gap to scoring the or
Compared
base
have Reference
alignment, exist
with in Sequence
be
amino
also
variants
specified [1], in Identity calculations:
or identical,"
aligned to sequence
compared
been identity the Sequence
the
the above matrix
in
being and
amino a
according
acids
claimed
Sequence, which Reference Compared Reference
Sequence
applied identity%, that
then
analyzed
and
base counted is values.
Percent
acid to
when in
about
may
(the (the and
the
For the
the the the
or to or in to
2010202722 29 Jun 2010 10 20 30 25 15 5
weak residues DNA the 3:66-70. acid has [2] matrices the used matrices synthetically common residues, conditions an which wherein activity semi-purified determining or luminescence, assays. may levels,
States,
agent other corresponding
shown products
database
sequence use in As
protein
The ones a) In
amounts,
relation of
from
from the
for that used one
contacting whole sequence polymer, Common endonucleases,
D.J.,
[3] supporting that HFE2A
instant
affect which synthesized.
HFE2A activity.
produced Henikoff,
protein modulates similarities. aspect,
an proteins, searches
the
herein, Gish, radioactivity, comparison to cells,
information
genes.
or such
polypeptides, BLOSUM-62
the invention especially and
forms
polypeptide
Alternatively, a assays blocks."
W.
the
stability. an
cell
test the
alignment
S. by or activity.
using
are & the
activity These
present
a extracts
or Altschul, & they
[1] treatment terms compound
in
subset activity
useful theoretic or Proc.
provides
use Henikoff,
any application-specific where Altschul, [2].
Polypeptide
matrix other may In may of refer is
"portion," score
invention
batch
Natl. terms
attached
or said stretch The measurements
S.F. of for of
include
of be this
reconstituted 32 to
measures perspective." an a with [3]
numerous
J.G.
Acad.
the
has polypeptide; number
S.F. (1991)
procedures said larger a
larger
of
is HFE2A
is
continuous
of
an "segment," formatting relates among testing
to a
assays (1992) exonic
part (1991)
polynucleotides Sci.
positive
polynucleotides a
HFE2A sequence. "Improved
scale of solid
of
polypeptide, scoring
of USA
assays
positives the
to cell
J. of
protein based and may "Amino and may "Amino
a
support, a tissue
sequence Mol.
value. the
compounds best polypeptide
89:10915-10919. and column extracts,
method
intronic
also matrices."
sensitivity
Such
which also assays,
Biol.
or for
on "fragment,"
is
acid acid or
Experimentation
with
be comprising:
gene such the detecting that
include
procedure terms whole for fluorescence, 219:555-565. sequences of or
used. measure
substitution
substitution
the
any
number
and to nucleotide purified Identifying of as could transcript Methods
nucleic include identify assays
a
animal of
when under
those most resin
the
be for an of or of
2010202722 29 Jun 2010 10 20 25 15 30 5
agent result cells of preferably 1 the such heart expression engineering, contemplates including as but polypeptide preferably polypeptide polypeptide herein. insertions polypeptide disease is step in
-9.
the activity a
use
cell
decrease
wherein
embodiment,
of (b). cells of that bone wherein b) In In In
In of has
said of greatest
a determining other by a
one polyethylene is a modulates
and
at (especially In
preferred iron
of preferred
marrow, been
genetic mammalian that the absent having
into such
contacting, least
a
embodiments the a
or
preferred preferred the a metabolism. preferred gene
result
reacts an engineered the change interest
as
polypeptide 98% said
details
engineering, an said although
increase
embodiment,
where embodiment, the a contained
atrial membrane of
glycol, change
amino cell
with
cell, identical
embodiments,
binding
engineering. activity embodiment
embodiment, in include
of
cells),
in has
the
and
the
an to
cells
in such which viruses, acid
in is contain,
in been
activity
activity, antibody to of especially which activity to,
said pancreatic said or
the from macrophages,
present the an
said
procedures sequence the the
cytoplasm engineered
cell.
33 Thus,
HFE2A
and activity
of
gene observed
the may of other
such or cell sequence polypeptide most
identifies
that
such a
have Methods where the
polypeptide
due
is
polypeptide be
has cell, tissues the
agents
engineered of
preferably polypeptide.
at reacts like, a
need method, on
said the of to
change recombinant to present skeletal least
hepatocytes, the
of
said
its
are
well comprise the by physical
sequence may
SEQ are cell HFE2A with, not surface,
95%
is said available
test is
known
cell
wherein
the does in be muscle
useful part by invention
be
to ID
or
activity
chemical
compound employed. other
identical
be
and polypeptide further
said polypeptide cell.
NO: insertion
is of said not of intestinal
in
enhanced
for specific
to an
cell
SEQ
said
the than
polypeptide, not For possess
10-12
polypeptide
in effect specifically
intact treating
described
and
to,
agent art,
step
HFE2A,
change
through
ID genetic of
as
In
for,
cells,
more as
such such cells
NO: cell,
is one
and and the (b) the an
of a a a a
2010202722 29 Jun 2010 30 15 25 20 10 5
where the to acid various compound compounds techniques. and experiments, immunoprecipitation, known receptors. Polypeptide) Compound techniques. transiently quenching useful compound, expressing of assays, plasmon means lipid assays, polarization, Polypeptide
changes
non-limiting amino
yeast substitutions. modification
any The Agents compounds
including dyes In
cAMP second
resonance,
acid
difference two-hybrid Such
metabolically
the
screening
in
and a screening
HFE2A Indirect are
Compounds with to
to
co-purification and assay
wild-type
confocal
sequence that detection,
example, change art,
interacting
also the modulate
such messenger but
In
in
known
chromophores,
have
interactions half-life conditions an in
including, the not useful Polypeptide screening.
fluorescence technology
as
use amino assays
especially color,
labeled HFE2A of
present fluorescence limited sphingomyelinase, nitric
that
an the
SEQ
the proteins
or for
of of
by
reporting acid
assay
or
modulate oxide effect can
the treating
resulting
discovered for
ID during
interaction to extraction epitope-tagged Polypeptide Such
may
invention. fluorescent
such
sequence
preferred
assays NO:
direct polypeptide with example, can also
for synthase, 34 radiolabelled of also
interactions
translation, 10 such
diseases
as
imaging be from
this fluorophores the modulating be detection
-12.
be (but and
identified of
embodiment,
of
interacting is
inositol signaling/quenching
based as,
wherein half-life the
kind
monitored due
radioimmunoprecipitation,
phosphodiesterase the the is
chromatography not
of
G-protein constructs activity
determined or
contacted can
only
of
methods iron triphosphate
target
upon limited invention
proteins,
by
the of
agent
such or
scintillation be to
a metabolism.
proteins
the through of
the
fluorochromes conservative half-life
further variety
the
the
coupled peptide
to)
polypeptides such with
polypeptide comprises
polypeptide
in using
ability may
polypeptides. the
fluorescence
assays.
in
techniques, assayed cell a of pull
or as of
activity,
candidate
response ability
proximity methods standard
(HFE2A receptor
employ surface viability By HFE2A of protein amino
down
cells way test has and
co are are
by of or
2010202722 29 Jun 2010 25 20 10 30 15 5
disclosed (or added, Such allow agarose bound polypeptide). compound polypeptide protein-protein which then to employed immobilized may the with alternative compound spectrometer. specific high-throughput Pharmaceuticals, also protein (which specific HFE2A
HFE2A
a protein.
be
be interactions
collected,
conditions proteins fragment
In
can
to are
binding and
non-specifically
binding employed binding Polypeptide
column
one herein
a
in successfully embodiment, under
presumably
screening masses is allowed suitable
polypeptide an
Binding
then A such associated
interactions.
assays,
are thereof
of capacity. and automated
may are data in between conditions Inc.
to the
washed
separated
time
included the assay
support useful,
modulating
bound to identify (Cambridge, include analysis embodiment,
test
HFE2A
which displace
associated or the compounds
case
to can
with
In
HFE2A
compounds an
for ligand to If
support associate
the
(i.e. ligands this
test
(e.g.,
desired, in use use from
then remove
of, epitope-tagged the
Polypeptide system which
purified interacting the agents case
buffers,
compounds
purified identification of
for
MA) with
polypeptide the
nitrocellulose Polypeptide
be is
such can
are original 35
buffers
with a
the proteins
other example,
preferably mixture.
the
correlates to
it used
pool or
of be
the
temperatures, released
the or
as
polypeptides the semi-purified bound
proteins interacting
the
tested
proteins
that semi-purified of
polypeptide test
with that for protein.
form
or polypeptide. to
system, test HFE2A/ligand invention.
and
a
or done
minimize
other
HFE2A mass multiple remain compound specific for provided
and his-tagged
compounds are
an or other (e.g.,
their
Compounds
proteins).
under
fragment
cell with antibody thereby etc.) measured
data encoded HFE2A
or
Polypeptide
associated
target
Other
binding
a
bound purposes. ability
interference The
the constituents
low,
by
cell
which conditions with mixture. complexes
form
support.
are
immobilized identified Neogenesis Compounds polypeptide
protein,
well or
to lysate) thereof) medium
by molecules the affinity
with
by exposed promote a interfere
of
with
genes will known metal list In
mass
In said non
with The that that
can are
are
for be an as or it. is of a
2010202722 29 Jun 2010 20 30 25 10 15 5
the other alternatively, support, interacting of employed electrophoresis, isoelectric phospho-amino releasing with the molecule assay purified together different polynucleotide. physically patterns label
the
presence polypeptide
an
to
cell In This protein 4. 3. 2. 1 can
interaction it; polypeptide .
when and but Collect fluorescent an
Harvest that the
Label
Expose in separate focusing. constituents assay
proteins, be
alternative fluoresce
these
associated, proteins
or latter
can is they
performed
absence a fluorescent an acid known
Western,
the
may
the be given between
assays
polypeptide are is
(i.e., interacting
Normal
label.
the
used encoded bound when analysis, employed,
interacting
include
embodiment in
to can
factor proteins
of dyes
close
immobilized
interact using
blotting to
to
the
a Use
they readout be
to and
gain compound help
the
that two; polypeptide is from it
protein proximity protease
analyzed,
are
dyes comparison a
are mutant are molecule
following binding
with additional
distinguish
and
purified quench and
data. designed not
the 36
released released
that
a polypeptide
(or in
detection
being support.
to forms polypeptide
digestion, to.
close for
steps.
and to will other or each each
information of
In
the true to
tested example,
(for
semipurified from
the
couple
produce of addition, proximity);
identify
other
The other
immobilized molecule)
binding such with example,
normal
is
the
protein for
encoded
released
versus a dissociated
when
specific HFE2A polypeptide its about polypeptide by different suitable when
proteins. and
ability
protein SDS-PAGE
with sequencing, by
they
when
polypeptide
by which
mutant
proteins
incompletely heating), Polypeptide
antibodies, fluorescent to an
a quenching
are
from
Such or
they interfere
second, without can HFE2A part
forms other close
and
gel are
or, be an
its or of in
2010202722 29 Jun 2010 10 20 25 15 30 5
thereof follows. to for Resonance acceptor labeled a to medium biological determine binding domains, entirety. ability include cell proteins may Nature ligand). patent
given
it;
inhibit its
function find
ability
An 2. 4. 3. The Quenching 1. One to Cell
and
publication Genet. polypeptide case,
cell
(i.e.
or (e.g.,
Measure may or
The influence other Based Label
activities alternative
HFE2A Provide
Expose
description function
high-throughput useful
inhibition couple Energy enhance
growth to
assays which depending
be hepcidin
interfere rhodamine); 33:
ligands.
an
developed on
and
biological
fluorescence
21-22) may
a axonal
the the Transfer WO assays, of
assays in
interacting is suitable HFE2A which the
or assay HFE2A
of
FRET the
with encoded protein dependent
acceptor-labeled
be Assays
02/051438, the on enhancement
presence may growth, presence
can screening
involved such
which either activity an
which (FRET)
HFE2A for assays binding FRET
that
be
interaction (OMIM:
resonance
be based
protein such protein as
a
pair in may inhibit
measure designed.
that on
donor
ligand where are or provided of assay. with incorporated in assays
37
assays.
protein
of
on HFE2A
absence 606464;
be works
cell-cell RGD by
related.
(or fluorophores or a
interacting or
between
whole
(e.g.,.
energy of
useful compounds specific
test
a This the enhance
such other
HFE2A.
herein and for
In interaction suitable
activity
adhesion. compounds. of
capacity See nitro-benzoxadiazole
cells, these
Either
assay for a
transfer.
as
herein
the a ligand. von variety
molecule)
teaches
the molecule
compound
those
is
HFE2A Those Roetto,
cell two;
assays, are one polypeptide
can can
Willibrand used
development of
is by
extracts
of
An evaluated
and
described can
be a
be
the
a
assays
skilled
reference
in
Such
test A. binding wide
with to
assay
performed
a
the be measured
being
Fluorescent et measurable
the
compound
or
variety applied
factor-like assay. fragment
functions a al. in
is
for
(NBD)) purified can donor- in of
the with tested
FRET ligand 2003.
in
PCT their low,
as
art
be of its in to a
2010202722 29 Jun 2010 25 20 10 30 15 5
test that designed and duodenal calibrated. a modulate iron a macrophages J 59Fe-bound possible the found 02/051438), 2001 Blood, may from proteins similar a systemically protein (RGM), 14;275(2):1023-9; epithelial peptide. molecule
CaCo2 biological Nutr putative
compounds measures
transport
may diet. cell be
in In Apr
Vol.
must
structural 1996 (fig.3)
neural suitable functions
involved One membrane. cells, these other which crypt This interacts
be membrane
extracellular 23;1 92
activity
For
though
as transferrin be
adapted to
Sep;126(9):2151-8;
and behavior
a
proposed No. interaction
like cells.
cells.
a
538(2-3):242-51). induced will cell relies transferrin. considered test
for example cleaved
in domains
to or
of 6 with
of hepcidin,
assignment reduce
compound
(September This based
adaptation Alternatively, chicken
In
neural HFE2A
Feeney HFE2A. on
include as
uptake in any
proteins responses cleavage
mechanism
the could
can soluble an
iron
Or, receptors, doubtful, case, HFE2A
with
assays,
development
and as RGM-like biological
assay.
GP
be
Signaling ’
in iron HFE2A s
be
uptake. of
on include a
ability
15),
modulates adapted protein. human the
related
Tandy and at HFE2A site HFE2A direct,
signaling to
the uptake
38 as least may One
HFE2A an of 1998:
human
may
Worwood suggesting to molecule
activity initiating
Similarly,
basal-lateral protein
assays
those action
hepatoma at
proteins
HFE2A modulate
S, Like to may some may
and
shares
modulate
modulate
the pp. in
the molecule.
assay
et and
the
repulsive
an
of described induce
immune assay basal 2157-2163; and
could
can apical al.
of
(fig5).
test
thereby M.
intracellular HFE2A are protein chicken
HFE-CHO
that these
sequence adhesion. in
J
cells mRNA
be
surface
transport
compounds surface
96-well
known Biochim for Biol a be release
surface
Iron RGMs
developed it
change
response
guidance
functions (HLF). find
induced
compounds in that acts
RGM, response
Chem expression transport
Nunez
Su,
to
of
of plates
cell a
are similarity of
uptake Biophys
this signals,
as act
the modulator of the
Assays in
HFE2A iron M.A.
may
a model adhesion, 2000
to MT, based
NRAMP2
a (see locally
molecule
intestinal intestinal iron secreted family
wherein
assays is
HFE2A
across soluble
of which
inhibit
et is
et
Acta
first that
WO and Jan
and iron has
not for
on by
al. al. on of of
2010202722 29 Jun 2010 25 20 15 10 30 5
These A to cell induced induced then Alternatively, on inserts, and cells. grown A cells, in lateral) B, side transport, secreted Caco2 "donor metabolism, secretion measure into direct cells, HepG2
mammalian response measure chamber mimicking
the
the line of tight
express
or
or respectively.
An ”
Whether on cells
cells,
the chamber membrane
Caco2
cells or B iron
via
indirect response
and
protein,
the example
of junctions.
an chamber, iron
cell RAW
active grown
an
can
to
transport "acceptor
and transport containing iron bottom cultured
considered
HFE2A HFE2A
cells cells
is upstream HFE2A
uptake
effect HF2A
of
be cells, exposed
this
likely
to transport to
of and the
(i.e. These
and
become
within
ofthe monitored. Grown confluence the a
”
assay. multicellular
can
from cells
protein.
CaCo2 compound
induced of acts
exposed human express can HFE2A the from an
interaction B
the
the
to
be two
millimeters
B
of the
HFE2A can purified chamber.
be
as polarized
on solutes chamber conditioned assayed
the Caco2 standard
iron assay.
cell liver
intestinal
can induced a Appropriate form The response. HFE2A. permeable be to
screening
A through receptor, communication
the types
transfected protein
resulting
biological now carcinoma
in cells 39
transfected distinct chamber,
with from Recombinant in
This surface,
the
B for
a by
lumen
be
media, The
chamber are are cell
the the A
the directly
measuring The cell assay adding
membranes applied
intracellular cells
chamber. apical in
a
culture media
known
intestinal
basalatoral activity)
thus
basal through
cells
modulating with human
iron communication and
assay
donor
for which
system
added
solutes, and HFE2A directly HFE2A
to
iron
or HFE2A from surface system. absorption
to
transfection
the
This
intestinal
colorectal is
the
epithelium basalatoral mouse
cells may relies be
absorption signal
in
to surface
a can
these B
iron
can
releasing cell
system to while
Transwell involved the
Caco2 chamber of constructs
could on measure
the be
to uptake.
be the macrophage
molecule system B
absorption.
transfected and/or
an carcinoma
the
the of applied would from
chamber. produced
B
surfaces is acceptor
from
also
the HFE2A HFE2A
HFE2A
in
(baso- blood. useful apical of
plate
This
A
and
iron cell iron can cell the the
be be to or to
Ο CM thereafter be applied to primary drug compound screening for iron modulation in intestinal absorption. Jun
Using whole cells or cell extracts, assays can be developed for
29 compounds which increase or decrease GPI cleavage or secretion of HFE2A 5 or for compounds which increase or decrease N-glycosylation of HFE2A.
Assays that measure compounds which modulate post-translational modifiers of HFE2A can be used to identify potential therapeutic agents. Some assays preferably employ purified or semi-purified HFE2A protein assays. This protein may be the GPI-linked form, or the cleaved, 10 soluble form. I125 labelled HFE2A may be useful for these assays. Such 2010202722 assays include aggregation, assays which are designed based on the tendency of HFE2A to aggregate or homodimerize via the RGD or von Willibrand factor-like domains. In this assay, compounds are tested for their ability to enhance or prevent aggregation of homophilic dimerization domains. 15 In this case, the protein-protein interaction assays above can use two pools of HFE2A, each labelled with a different fluorophore. In some embodiments a GPI-cleaved form of HFE2A may be used with the GPI-linked form. An alternate semi-purified format includes yeast 2-hybrid and/or phage display formats, wherein HFE2A binding regions are incorporated into both bait and 20 prey vectors. This format would provide a moderately high throughput screening assay, suitable for radioimmunoassay or plate format.. In addition it is to be noted that HFE2A may interact with other known proteins of the iron metabolic pathway: Transferrin, Tf receptor, Hfe, hepcidin, p97 or other iron transporters, and receptors thereof. This interaction is a 25 useful activity which may be used as the basis for a screening assay. Additionally, drug screening assays can also be based upon polypeptide functions deduced upon antisense interference with the gene function. Intracellular localization of HFE2A, or effects which occur upon a change in intracellular localization of such proteins, can also be used as an 30 assay for drug screening.
40 2010202722 29 Jun 2010 20 30 25 10 15 5
These decrease) contributes agent in transcription embodiment, cell, result polynucleotide found intestinal be (CAT), amount polynucleotide Green region In compounds Thus, sequence amino hemochromatosis. humans)
one preferred measured
most
of
thereby Another in a) In Such b) that agents In of acid and assays
aspect
Fluorescent of
said
tissues accordance preferred
HFE2A
cells, and
detecting contacting
preferably transcription derived
a
the to
sequence modulates
for modulation
embodiments, transcription
is
contacting; the
or
amelioration
that
which or like). corresponding seek the broad conveniently evaluation identifying
hematopoietic monitored
of
promoter gene is
from
embodiments,
interact the
present In
operably a
to
a Protein,
under with is
category of change
addition,
activity.
cell the identify human of is individuals associated
a
product.
preferably
the as
of the has protein, is
an with selected such activity physiological invention
measured
therapeutic a present
linked
to
HFE2A
in
cells, luciferase, foregoing, the disease body
agent compounds
of several
the hemojuvelin the
transcription
the
sequence screening
designated In found
to of
promoter
with a
from (for pancreatic
transcription
in
41
a polynucleotide relates decrease
gene. that a
a of (for an
convenient
mutations,
reporter
agents example,
hereditary polynucleotide conditions to the iron among
chloramphenicol
intact
example, modulates which
have
Relating
of
assays or region to present metabolism, is hemojuvelin
SEQ
or cell, of
HFE2A cell, a measured
gene,
macrophages,
of juvenile
an the modulate SEQ method embodiment,
have
transmission
of
a
said ID skeletal whose
preferably is increase to reporter
invention. invention
chemical
HFE2A
that
NO:
transcription transcription transcription ID
represent whose
polynucleotide been
hemochromatosis. comprising:
or
NO:
for by
transcription acetyl-transferase is,
19); (i.e.
muscle
HFE2A, genes
in measuring (in a
identifying
found
a
10-12 provides
agent the and hepatocytes, transcription. transcription
gene
increase mammalian a
of candidate
preferred
promoter juvenile
such assays. cell assays, of
in that with whose
from
as
said is
this and the
the
an
or as is to a a
2010202722 29 Jun 2010 25 20 10 30 15 5
transcription wherein other transcription genomic comprise embodiments, cells employed, having identified regions which of compound(s). ligated ID adenovirus, considered plasmid systems systems a employ can as polynucleotide protein, vitro
reporter being the No.
be
assays
cells from
can The In
been to
reporter and the 19 in
used,
the is
or such
involved
gene, a accordance said which
an
transfected
be
of or
invention therapeutic
as gene, genomic untranslated
reporter promoter cell of other engineered
for thereof.
adeno-associated appropriate
the
Those used 22
as as or HFE2A such
for polynucleotide,
does drug such
gene
polypeptide CHO may and a bone
in whose
tissues
example,
to
tool
gene,
HFE2A promoter an also
compounds
regions In
screening. not
as into
produce other is
with agents
are cells.
marrow, be
to
regions to one intact
constitutive,
cell
in express claims
expression
a
and do
express
then may
employed the
in assays cell, publicly
in
Naked
of as such
in so.
of
region
an cell incorporated such
large vitro,
HFE2A
the may virus, disclosed neurological,
Alternatively, disclosure cells recombinant
which
and Alternatively, modulators
the also animal
assay,
is
invention. a to DNA
or
or 42 of enough retrievable as
be
the
level subject
a protein,
of
can
can identify
include HFE2A
increase Gene bacterial be
included
by
cell can
recombinant
and tissues
herein.
the
be be is into
genetic
used. herein,
amounts
that be of gene
are CNS cells
DNA-liposome the cloned
induced. easily non-recombinant where upstream and compounds
sequence a
baculovirus, or used, the
In
readily The systems, transcription
expression has or
or engineered or decrease In link
plasmids
engineering,
upstream
gene/protein,
measured. into such
cell cell polynucleotide gene of spinal or
been additional it
obtained
protein genomic
to a
expression is
databases. lines
genes fragment
which disclosed
a
and exposed
cells, complexes the comprising engineered
construct gene,
herpes
plasmid. to
untranslated
cells
to where
expression Expression
from preferably
eucaryotic express
encode preferred modulate region although use
and
such
vectors can
absent thereof
to herein
Such virus,
SEQ
in may The test
the the can are
be
to
as is in a a
2010202722 29 Jun 2010 10 25 20 30 15 5
the treatment the genes. gene also analysis analyzing heterologous produced, properties. for response comprises gene NO: thereof. the therapeutic containing capable protein, on binding agonize identification Compounds humans)
other identifying
levels activity physiological
be 1-9 corresponding after
Such Target Recombinant Thus,
In
used.
or if assays or of
genes
encoded this
of
the
state from by
first The a
highly of
antagonize
producing RT-PCR, contacting of the
agents. in that recombinant HFE2A purified one
way, of
biological levels
an selectivity
the The
one
identifying levels
humans,
and/or individual may
chemical
to purified interact
of agent
thereby, response
the
HFE2A such
invention
cell
a identify
gene. to of
The protein
be of or,
recombinant polypeptides. number the gene
said
transcription HFE2A,
lines gene
that activity preferred or
proteins. determined
alternatively, are with
is such
transcription cells agents,
present with Polypeptide
on
transcription
cells an compounds
thus of assay are modulates,
therapeutic transcription
the
of the
agent high may of preferably the important also
methods
agent. embodiment, claims These
with activity
especially cell
polypeptides invention is
specificity
of
43
cells
before, and preferred be
desired. by of lines (such HFE2A
agents
can
preferably samples
recombinant agents
HFE2A
which used then
of
measuring can known to having aspect
a and
serve and
as small
specifically
the and
be
polypeptide testing
for the Those Gene
that in
of interact
Gene, encoded SEQ extracting
in
quantified can
the at
the
that
as
methods agent. the transcription
of the inhibits, organic
may
the
various
a cell or be preparation
such sequence skilled
ID
have the invention, as art,
marker,
a
used
with amount
NO: lines
contemplates have Accordingly, thereby defined
encoded
suitable
or
development by agent protein disclosed expression little
molecules,
points in
by
in the Northern
10-12
containing
indicative
assays therapeutic the
or a
of
or measuring
of of
for
herein,
variety
or
fractions proteins. no
reporter thereby, SEQ analogs purified
art during, protein
effects herein
other effect
from of blot this that
are
the for
of
ID or of of a a
2010202722 29 Jun 2010 10 30 25 20 15 5
a on through the which no preferably which testing identifying to is active computational treatment for metabolism efficacy. invention include hepcidin administering, assay Hpx body metabolism.
gene
those sometimes
effect. expression identifying crystal
mice
can is iron
or In Those Potential a) b) site,
thereby
the
or anemic
skilled useful
another screening
levels,
x-ray Thus,
are
(hypotransferrinemic administering
test not detecting with a
polypeptide content, structure probability
thereby compound that
usually a skilled disturbed referred
or
tens
for modeling,
in
therapeutic a in identifying therapeutic
crystallography mice,
radioactive
activity aspect, may the
potential modulating a
method
of
in further identifying
anemia in of art.
tested
millions
of
iron be to
said
of the with by to
an
of
the
test
as identifying
the
modulated an
therapeutic
at
as agent,
active therapeutic overload an aspect, art animal
compounds in iron
important invention
in
indices.
animal the least of invention.
compounds
disclosed
mouse),
animal
a are agent
silico
computer
uptake
or
activity compound, comprising: site
one
a the aware
an
a other mice,
44
change
screening.
in intervention, Specialized model
for provides
of
physiological
and present
other
agent) compound The agent herein,
in animal identified of
a generated to
that the
such the others. hemochromatosis
HFE2A
techniques);
method
systems interact gene
or in
found
treatment include typical
a gut, invention
and iron models
a its
method
Sophisticated
polypeptide. using and
mouse or
which
analog,
Polypeptide liver
to involves consequences compounds, metabolism
with
polypeptide, to
measurements demonstrating have transferrin
confirm (i.e.,
the relates
iron
for a the interacts and models
as
disease activity
computationally
methods (a)
content, mice as target
This a
(b)
software to
determining
protein the
due is a
compound
saturation, preferably
a
for available
with (hfe/hfe), using that
result
process through putative
protein,
method
of little to
of of whole
study
said (i.e.
are iron iron the
the
for
an or of
2010202722 29 Jun 2010 30 20 25 10 15 5
'
as assay administering a the functional a antibodies, substances diseases identification can with transcription, according polynucleotides gene agent trails also expression Monitoring Polypeptide Gene, example, inadequate by
condition wide
a
a transcription
patient
high be
in
of transcription,
screening method determined The The
In as
clinical variety
subjects monitored
a
of
the homology well
to
further
the including in
studies, screening afflicted iron
or
gene the which invention to
and protein an
individuals of or
as
trials. of
influence said
metabolism. biological the invention or disclosed assay animal
exhibiting
aspect, structurally purposes,
development by
epitopes
activity transcription, in protein with target, thereto, animal invention. monoclonal
drug levels, For
a clinical assays
screening as a
afflicted also
studied
of the example,
disease
can
of
an levels, activity described symptoms
screening herein interact
or agents and
including
the present
and thereof. trials effective Preferably, be of
includes
biological
with
of
antibodies. herein. genes including
the applied
of protein functionally
or
assay
the can therapeutic
of of
(e.g., iron
with
a
invention
biological 45
invention
herein
but of amount
subjects disease
effectiveness assays,
the
or
metabolism.
Alternatively,
be
to said not diseases activity antibodies
small Polypeptides or levels, not polypeptides protein
modulate Such
HFE2A only used
to animal
related bind
of relates
limited
of
activity.
thus agents exhibiting
organic
increase
an can and/or
in antibodies iron
levels, or of
as
to agent basic
is
simplify
of
be to
genes, polypeptides
iron transcription to, and the biological metabolism
a
an
hemojuvelin,
for disclosed In
an a
compounds) monitored human
or diagnostic
therapeutic
encoded
drug first effectiveness method metabolism
or such
decreased agent
antigen the
biological immuno-reactive
will including
the decrease identified
screening,
treatment clinical patient, be
activity
determined herein evaluation,
for
comprising
of
useful
in identified to
by
treating
agents, agents. HFE2A HFE2A
on altered
activity clinical
due
genes
by trials, of
raise gene such
and,
the (for the
but for an
an of to
2010202722 29 Jun 2010 20 10 30 25 15 5
following disclosed treatment described small agonist, monitoring preferably, metabolism, the effective with a polypeptide, thereof, or sample of may transcript generating administration mRNA, and proteins iron persons non-recombinant
decrease more expression
subject
iron
be other metabolism. molecule, Where In Specific
Purified
or or
desirable are of antagonist, in corresponding metabolism. a
said
amount
levels
for herein) genomic
samples,
preferred herein; the the
such
nucleic need other
accordingly. themselves can i.e., in
the
administration an
or
genes effectiveness sample compounds
gene
in or
or agents, be
to
activity thereof forms
patient iron of
including genes to Those
a
increase
(iii) other DNA
semi-purified used acid
thereby
peptidomimetic, embodiment, cell an
decrease
transcription, disclosed
metabolism
of
with
ascertaining to
agent therapeutic
and of
For of post-administration is to compositions for that drug
skilled
these
HFE2A, HFE2A non-human, the
which ascertain said
the showing and of for the example, that
have
the identified candidate steps treatment
herein
effectiveness determining
proteins protein, (vi) treatment
in
the
HFE2A will
of
disease expression include such
and
the agents altering
the protein,
been the
of 46 the present
an
modulate and
increased
which
biopsy
(i)
any art
effects mRNA, effectiveness using or
as of
identified improvement
corresponding
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concomitant
of implicated
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the conditions samples are by
biochemically
peptide, of
invention
disorders,
subject the or specifically disorder; samples
one
the of the administration measuring
familiar or activity
administration
invention.
drug
or genomic agent. by of gene
nucleic
and
with
antibody,
can
of
fragments that in,
the of the
provides
reduction administration can
(ii) in such
of a with administration. comparing post
expression
for
hybridize be an
particular
screening screening
a modified
gene levels
administering be iron
acids,
DNA
patient Recombinant
administered agent
as example, techniques of
taken administration
nucleic a of
the
metabolism or in diseases
method thereof,
of in the ribozymes
problems
encoded (e.g.,
drug. the
agent requires with versions the to or
protein, assays assays
on
agent
show acid,
gene level
iron pre
one
for the an an
for to or or of to
2010202722 29 Jun 2010 20 30 25 15 10 5
treating are and HFE2A compounds specific antibodies antibodies manufacturing CA), provided antibodies which those of art, include Protein etc.). such occurring, it sequence be mimetics instant include herein. Polypeptide
is
HFE2A
confirmed
gene the
taken
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have Specific
Design specification, the gene like). gene
other
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small others. therapy
found
by
or
or in (polyclonal which tend up
diseases
are for
the protein
the mutant in
Abgenix,
they These
(including, therapy embodiments, as
and of
compounds
the compounds Labs,
compounds well to organic to treating
desired
body
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useful specifically allows
are
may
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in specific
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previously
are
Inc. or widely can effective compounds and vectors such preferred, a Inc. be
exons in
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molecules. the
compounds
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synthetic.
be
are animal which to which
(Freemont,
which sequence. cell. compounds
delivery treatment available
bind
those
diseases
easily useful thus comprising
or at discussed.
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modulate reducing modulate introns
are to models assay,
modulate
skilled changing
Collections
of
and 47 adapted for
an CA),
Such well
from of
with as As
HFE2A CA),
vectors
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epitope
of
modified the
such those is
known
all (for Genentech
in Design
disorders
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the
commercial
compounds well
such therapeutic compounds ability.
the
the Medarex, compound the or
invention,
to
example,
as in activity
activity thereby
and
generated
art. known
a of to
identify activity an
level genes, the
and antibodies
part
Agonists, those previously.
said
combinatorial In organism of
assays
(South
manufacturing of of
particular, or
to
Inc. iron
modulate sources. of of indications
of
mice, and
polypeptide.
HFE2A
of promoters, HFE2A therapeutic skilled may
those
amount
the
the the
using
metabolism
(Princeton,
HFE2A or disclosed San
antagonists, are
invention, to invention chimpanzees,
HFE2A
fragments Design be skilled
in
As
cells in
humanized in techniques
the
Francisco, libraries useful
of the
disclosed the
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naturally
know
of 3'-tails, include HFE2A
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These agents
where
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in in gene such
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NJ), and and may may
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for
of or to of
2010202722 29 Jun 2010 10 25 20 30 15 5
•
demonstrated from compound expression extract even be the semi-synthetic) having HPLC molecules to Lead to, for understand of (Merrimack, compound synthesis) as compounds exemplary animal-based number Biotics extracts Institute
modification the combined saccharide-, natural generating art.
libraries, synergistic, Optimization
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screening (Sussex,
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are (Ft. general, supernatant
FPLC)
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NH)
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UK), until
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Aldrich
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FL), Xenova fermentation
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commercially
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gene of
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available and
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Development
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PharmaMar,
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limited of compounds
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the or
nucleic
broths, cells). agents libraries
synthesis be
test of 48
of from
(Milwaukee, drug of compounds,
Numerous therapeutic
invention. available bacterial, both UK),
purified extracts
a
to,
activity
In
Examples smaller
or acid-based and to a mixture
discovery
purification
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according
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plant-,
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minimal number achieve mixed
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from methods fungal,
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subsets
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expression
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compounds to Alternatively,
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methods sources, such plant,
are development of
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extracts
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2010202722 29 Jun 2010 20 10 30 25 15 5
fractionation fractionation according addition, detection characterization extraction, constituent and readily may replicates developed from active agents combinatorial chemically compounds having biodistribution, active selecting standard identified purification In improved
a
chemical
preferred be a
component When De-replication(e.g.,
In Improved modified
core large such which employed
natural
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as characteristics either of general, or
to
of modified
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methods.
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methods de-replication, are
embodiment, means) such
pharmacokinetics
crude
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tested
whenever and not
of to these
heterogeneous identification of synthetically
positive according
therapeutic standard
directly be extract to
in which
of
Furthermore, materials known
(i.e.
product
activities.
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for to may mixtures useful
an taxonomic screening
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the
a possible.
or and
identify iron
derivatives
the
are
is also related chemical lead
chemical, in
any
improved agents to
found
extracts
metabolism already
value further analogs of produced or the
observed
The
include of
purification methods combination
extracts
extract
a
other
if de-replication, 49 those to methods compounds
chemical art,
same to agents. desired, for are library,
thereof. modulation
known evaluated physical,
or
analog
have
compounds
of
the e.g.,
desirable
subsequently libraries
is
synthetic are preferred effect.
disorder assays known
such
treatment
for possible
entity provide
therapeutic
for thereof) any by known Methods of process
can or example,
their the
and
standard core of are biological
Thus, described
biochemical in
library features
within
known
compounds
with
the be
invention
analogs
the in
produced,
a therapeutic condensed to
or of
analyzed structures of
used
the
therapeutic
ready improved
the the is
pathogenicity the
activities, isolate one or fractionation art.
in
extraction for
de-replication, art. the
herein elimination compound
crude
the
to goal
produced which is Compounds methods.
therapeutic
means
if of art. If
effectively using purify
chemical
activities
to
desired,
desired,
may stability,
interest
careful extract of
further for
target. a
have
and
and
any few the the are
the
for be by of is
2010202722 29 Jun 2010 20 10 30 25 15 5
or Therapeutic delivered, biological systematically body clinical Although safety formulations Conventional more invention pharmaceutically-acceptable A.R. formulations administration formulations, intraventricular, subcutaneous, glycol, excipients, example, may intraperitoneal, polyoxyethyiene-polyoxypropylene biodegradable Formulations
further
be
expressing
Those Compounds Gennaro Methods of of oils
and in
for analogs activity the Remington:
either
the the
of sterile Agents
human oral use
or may
in skilled
pharmaceutical vegetable
new
for screened form
intranasal, may the assays well
intracapsular, compositions
in by and
AR.,
thereof, the lactide water, intramuscular, administration
be
treating and form first chemical parenteral
of physiological clinical known in
be those
The in
HFE2A
liquid the Uses
1995, identified
of of origin, the or
to employed, according
or
Science
powders, the
art in that saline,
trials
the polymer,
evaluate diluent, Thereof
form aerosol solutions
entities
the practice
to intraspinal, diseases are Polypeptide
or administration Mack
effectively
invention
administer art as
which means is
hydrogenated of
familiar polyalkylene copolymers
and
intracranial,
to
carrier, nasal
for administration. useful for 50 tablets and
preferred,
whether the may or Publishing
mentioned Practice making
lactide/glycolide
are
or suspension; example,
intrathecal, invention. do compounds drops,
with
protein.
be
therapeutic
or may assisted
such or
used so
excipient,
they
employed may,
the
may
glycols formulations capsules;
are napthalenes. of any or
be
intraorbital,
compositions herein. to
Company,
Pharmacy necessary aerosols.
Analog Therapeutic intravenous, then
modulate
be
means, for epidural, establish for appropriate administered
first
such agents
in used
therapeutic
oral to
example, and
unit
identified copolymer,
compounds provide
are as
.
to
Biocompatible, administration,
steps to
the (19th for
dosage using intracistemal, Easton,
efficacy
polyethylene formulations
found cells to ophthalmic,
control
parenteral,
intranasal
identified
route
patients.
for suitable ed.) agents,
with contain one by
of
in, form.
pre- and
are the the PA.
ed. the for or of or a
2010202722 29 Jun 2010 20 25 30 10 15 5
together therapeutic systems release therapeutic one conditions, administration ether, particles, may may Formulations situation of sufficient identification method code structure assay as herein) more numbers research information information having screen
said a
be
be of result
assay
numbers)
The Combination
(user
agent. glycocholate for wherein
for
administered the aqueous identified
for or of
whereby to purposes are or osmotic
contemporaneously.
compounds
agent of producing
as present
agonists such processes convey the agents to 1
disclosed identity of
for )
used
in said For to may
reproduce
such
said and, the
compounds.
structure, inhalation
identified solutions
as a example,
identification the pumps,
according the the
identified screen therapies
form
user invention
of of
product along and after product, where
a
of
the processes chemical first having the product, compound,
of the of
the
by dosage, invention performing deoxycholate, a containing,
with nasal may
the compounds implantable an
user
present is number therapeutic
according to one
are also
Other
comprising
the
assay agent the
present process,
another the contain such character drops,
is
of
also
for
desired data etc)
relates
simply
include invention.
the invention, 51 then
of
potentially
of as and
for the identifying
contemplated to invention or test
to
(such agent screening collected
the infusion excipients, or
by
and
example,
identifying another as or and/or screening
conveys
the given to enzyme administer
assay, ethylenevinyl compounds generating invention
a
a may intended then antitumor as For gel. assay
useful
process specifically samples systems, structure
with such
where methods user
example, and imparts polyoxyethylene-9-lauryl or
modulating
be
that an
process, ,
may by procedures
respect
example, it test
who wherein
to
an
without parenteral
agent
oily a acetate for agents the
that
treat information labeled
use and/or to
disclosed number
data and agent
then
contemplates
the
therapeutic
a inventors.
solutions
to using
comprises
according
the a
activity second
knowing said
to user lactose, liposomes.
copolymer
utilizes said
are coincident
properties
disclosed
with (i.e.,
assay facilitate delivery
of
data one
herein
of
agent given
code
and, (i.e., user said
the the the An the
for
or
to or or to is a a
2010202722 29 Jun 2010 20 10 15 25 30 5
transmission expression information gene; by (for (user method an for said indicating result conditions activity activity said polypeptide analysis pharmacogenomic prognosis
the
HFE2A
producing test contacting, example,
of (a) In present 2), (c) (b)
(a) In (c) (b) In Diagnostics
indicating of for
said compound HFE2A
another
accordance gene
producing a producing contacting a
determining determining said
promoting of verbally by
promoter producing
to activity
genetic
further of contacting,
onset
a
identify
invention.
modulating
test
the test information
compound, wherein
Gene modulating such
and
by
compounds, based compound test or
or construct code test
data embodiment, under a an
test
with
the
a
I (HFE2A, a embodiment, test severity a Pharmacogenomics
in
data compound, activity
wherein said change data
change
data compounds activity. on
with
the number from writing compound conditions
comprising: activity.
with
change
a
with comprising
with
based foregoing, change of of
hemojuvelin
respect user
said in kits respect
in said
onset,
respect the
or respect
activity comprising: the with
expression
shows
change 52
1 and on
supporting
with having
HFE2A
some in to
invention present
to to
a
of expression a the the
user
to methods. of an to the
change reporter modulation, gene) juvenile
the
shows
the the a
said present equivalent
polypeptide; HFE2A corresponding
HFE2A
2
particular invention
of
is expression modulation modulation gene
relates for
polypeptide
specifically said
in
modulation, gene of hemochromatosis,
This
the
an polypeptide
polypeptide invention
the
modulating and
reporter
relates
diagnosis, modulating HFE2A fashion, to
operably aspect determined
of of diagnostic of
results).
contemplated
as
HFE2A said
and relates to
an
gene
polypeptide
modulating
a and
relates a activity
sufficient
linked including
reporter result method activity HFE2A
genes
under gene
as to This adult and
to of of to a a
2010202722 29 Jun 2010 20 10 30 25 15 5
therapeutic wild-type onset disease use disease hemochromatosis. mutations acid skilled sequencing, from blood, known PCR to expression, (SEQ disease (Elisa, correlated mRNA activity, protein assays mutation
develop
of
the
based diagnostic
hemochromatosis,
ID
For Alternatively, Using
saliva, in A Gene Based
methods. HFE2A Radioimmunoassay
can (HFE2A of to associated
and mutations of the valuable which No.
(non-disease
with example, iron
classify disclosed agent
techniques
iron other amount be transcription,
art
the hybridization mutational 10 urine on
metabolism.
such
are
diagnosis
are
used - Polypeptide), methods, nucleic the
metabolism;
for 12) All
embodiment
biochemical,
or disclosed
suitable (mutated)
capable patients using nucleic
a
or instant
a
other such protein to disease.
herein,
patient
such associated) presence
analysis a acid
diagnose
to both
disorder the probing,
tissue analysis compounds
for
acid invention, and of
as
herein.
classify having based
sequences sequences
thus
such (i.e.
developing
protein use thus comparable of mismatch
chemical
analysis
the
can or provided
the
with of
pharmacogenomics).
the single determination sequences
analyses as can
or absence
confirming
53 patients like) iron those each invention
sequences
those animal
presence at
and be
(SEQ
disclosed
can amplification,
metabolism, numerous
and stranded
can by
risk and
serve performed skilled
kits. such mutant the
be
of
having
tissue
ID
diagnostic (SEQ absolute, be of
will
a
the
used
patient. Nos.
or
disclosed of
Techniques as as having
in normal employed in
be conformational
different
samples.
absence polypeptides the this
the or
ID antibody diagnosis a or
to 14,16, on
and
to
diagnostic
It
at in No.
as
identify art
amount invention, use assays also a a
or the
myriad
risk well
in small will
types disease
1
mutant
to and
the of based selection this
relates
include -
of
as also
of the
identify 9)
a
of of that 18), sample
diagnostic other of
tool having
biological invention analysis, both
mutation
and
juvenile
HFE2A be nucleic HFE2A HFE2A
HFE2A, assays of
to
those result
for DNA
of well able the the the iron the
of a a a
2010202722 29 Jun 2010 20 10 30 25 15 5
factor the metabolism. to identification such chance in patients metabolism. Not Assessment variability or hemochromatosis, particularly The relation subjects HFE mutations c.569C>A,p.A190D mutation: at manifest Mutation performed result
HFE2A severity identify H63D.
Hfe
all patient
mutations as and What A
in
gene. of to
at
of patient adult
enhanced in
This homozygous onset
in adult further (in HFE2A in these
of risk
HFE2a
on
aids
developing
of There follows Using clinical HFE2A developed adult of one
disease
mutation of
a
the onset was
there
of
HFE2A
risk mutation assessing
having in
or
mutations
which
hemochromatosis. the
are
hemochromatosis.
serum
found
HFE2A penetrance. is diagnosis both is
factors hemochromatosis.
is
and
teaching useful for an
is many
the a
adult early correlates gene
copies
considered
in WT Type disease MT to which
example ferritin
them
HFE
disclosed a gene. be disease lead
in gene risk onset onset
may
and
heterozygous
of identifying of for ones
gtccaaggagAttggcctcta mutations, In gtccaaggagCttggcctcta Sequence
to
levels, of factors
the only the
for
It mutations some
of
prognosis be 54 of Adult iron the
a
hemochromatosis. of will herein,
revealed A
hemochromatosis gene)
invention, the mild a Nucleic
loosely
informative
development metabolism
iron transferrin second remain
severe for
human hemochromatosis and
diagnostic mutation
they
for could indicates
diseases in
metabolism.
of classifying with acid
the HFE2A,
a
relatively
it phenotype,
diagnostic subjects remain
mutation
the
be
is or the
for receptor following based which
now
used benefit of protein a
risk
of
(SEQ (SEQ known which wherein Thus, (i.e. statistically
a
symptom
patients clinically iron
possible
homozygous in does on
disease to of This
displays
the
test
while
the saturation,
analysis ID
ID of predict a homozygous mutations metabolism, patients detection adult
known NO. Hfe a NO. not HFE2A Hfe1 was discovery
mutation at
free. in to
normal. greater
of
always gene). a
32) 31 risk
onset
onset
other
used gene wide take
then iron
risk )
will
for
or in in of of
2010202722 29 Jun 2010 20 10 25 15 5
developing types risk mutation), way transmitted 985, in treatment drug in to for treating treatments genotype allow Gene. may upon polymorphism
the affected respond
of
the
the
occur 1996; action) for
administration of
response developing wherein a) This b) having Pharmacogenomics This Thus
pharmacogenetic disorders selection prediction body
of performing performing
of and
to persons according hemochromatosis, invention as
Linder, in
the
pharmacogenomic by
an or
a a
determining or acts a
a
statistically particular a
individual to determining genetic individual
single
patient
SNP)
a
finding
disclosed drugs
of of disease
M. on (Eichelbaum,
of
a
a
therefore a to
agents
first
diagnostic W., therapeutic drugs factor patient's
in
conditions patient
having due
agent).
of is
conditions
promoter, based which deals higher assessment
Clin.
of
examined the
in mutation
to altering
(altered
iron because
(e.g., discloses
this
analysis genotype, altered
response
with a
presence
Chem.,
patients assay on M., risk
metabolism, polymorphism agents, transmitted
specification.
intronic, can the
drugs) clinically Clin. 55 of the
drug to
in
for of
drug on
developing
a determine genotype 43:254-266, be will can
way
to HFE2A
a
a
including and Exp. HFE2A, method particularly
metabolism).
differentiated.
known disease a for disposition
develop or
significant
drugs lead particular
comprising
as prevalence
Pharmacol. exonic
therapeutic
(e.g., Pharmacogenomics
classifies
single of of
the said risk
to prediction of act
the the
classifying
1997).
therapeutic
ability iron the
sequences
factor
disease. therapeutic an and on
hereditary
disease. Altered
factors individual
Genetic
of
metabolism, the
single tailoring Physiol.,
abnormal said
In of
or
polymorphisms (such of
body the
general,
a prophylactic altering drug side
patient
conditions agents nucleotide
of
variations patient
individual agent. (e.g.,
as
23:983- of (altered
HFE2A effects action allows
action
Hfe1 and drug
two
the
the as for at
2010202722 29 Jun 2010 25 20 30 10 15 5
designed with be potential Gene families gene. required protein Affected from the from CA3AL590452 consistent JH7, G. Greek of using hemochromatosis. based assembly 143293826
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evaluated.
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is genomic 25
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al. sequence
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to existing contains of
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Such
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have candidate
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efficacy agent. ofthe thus stratification
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hemochromatosis
have
to
collected consanguineous. and these this
chromosome
families and be Cells presence comprises 1q21
genetic
While Patients
define region including been useful sequences for region
Gene known contigs.
duplications,
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a
Example ten
JH3, gene
can potential reported
haplotypes the
and the in
was in
Dis. mutation or
can consanguinity.
compounds,
Greek
56
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6, new
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patients
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the
and for
and
markers clinical
between in estimate shared
9a-9d.
Only
new
the (Papanikolaou,
Table juvenile in in 13 treatment
to kits
with gene
(143293543-
JH7
the 12 six the
of
individuals one to
disease families
individuals
which trials
sequence
alleles
identified
Of 6).
markers.
patients different markers the juvenile DNA HFE2A HFE2A defined content
family,
these
of can size are
for
or by a is
2010202722 29 Jun 2010 20 10 15 5
596177 CA1AL CA3AL 355505 CA1AL 590452 DNo. D1S44 unaffected Table at 8 v.12.31.1) transversion Identification one ENST00000306561, members proband. present mutation probably European European (c.842T>C, homozygous
displayed least
child,
6. We The Mutations
6 in
of
identified The carried and sib in all and
of
any first cacttgagcccaggaatttg TGAGA gggaaatggtcccataattcct gtgtgctacaagtttgccgaat Forward (SEQ (SEQ GGTACTTAGCCTCGAAA (SEQ
second different P-I281T). 7
this
(c.959G>T,
in
is unaffected, markers
of
the
74 JH3
other 37 sequenced heterozygous
the
Mutations gene.
ID ID ID
in Greek
unique
Greek
10
genetic Primer was NO: NO: affected NO: homozygous affected
HFE2A, Ensembl (SEQ JH JH4-203
in Sequencing JH
control 57) 59) 55) the
probands
p.G320V). are in
control
haplotypes,
ID
one in families,
variation
linked JH4-203,
individuals is heterozygous NO:
for v.12.31.1, HFE2A is
(LOC1
chromosomes. homozygous
of
homozygous this haplotypes.
chromosomes.
61)
Interval. three or
was
This
SNP. 57 it 48738)
identified family
a from
was initially
November, from
coding T SNP
Affected
This for
gcttgaaactgggagttgga gactcactgcagccttgacc cgccctgccAATAT (SEQ Reverse
(SEQ (SEQ to G GTGTCACACAACTGGTT
members
families
for determined ENSG000001 families
for
GT this was limited within
C
SNP exons
After the
this
ID ID ID
transition 2002.
SNP. individuals (SEQ homozygous
Primer NO: NO: NO:
was
C.959T SNP. JH3 this
to JH5,
and sequencing (ENSE00001
those
60) 58) 56)
not This ID that exon
and 68509, not
Two JH6,
NO: GTT in
allele present
from
probands present
SNP this
the 4. is
siblings
JH7, CT 62) in
a transcript
all The
JH4 and
the 277351, 3' was SNP
G
in
family JH10,
in
exon
to 128 and
JH3 that first one and
not
74 is T
2010202722 29 Jun 2010 10 20 25 15 5
Additional the this and p.C361fsX366) proband. in an (Ensembl) LOC1 provided SEQ
5
ensembl
JH1 specification: unique
heterozygous ID 48738 A To Exons SEQ Start:142000393, SEQ Start: SEQ Start: SEQ SEQ Start: SEQ SEQ Exons
1
NOs
below proband,
third
information simplify and
amino
ID ID prediction ID ID ID ID ID Exons
142002394, 142002394, 142001790,
for 1, 1,
NO. NO. NO. NO. NO. NO. NO.
was REFSEQ
based 3a, 3a, these variation,
acids
indicating
2 4 8 7 9 3 1 3b 4, sequence in identified
—
- - -
- - -
about translation_start: based are:
Exon2 Exonl on
Exon3b Exon3a and
the Transcript Transcript Transcript
and
NM_1
End:142000541 the
End: End: End: 4 JH11 a
the
that
a on
may
in NCBI
premature
deletion
searching, 45277.1 ENSE00001 ENSE00001
the 142002953 142002430 the 142001993 sequences
this
ENSE00001155188 4 2 3 also proband
homozygous ENST00000317920 Exons sequence Exons 31
individual 58
be assembly 208 (NCBI).
at
stop. found
Chr:
1
exon 1,
277320 155182 disclosed and , Chr:1
Chr:1 Chr: nucleotide 2, 3b,
AK098165.1.
1 is This
3b,
Exon at
state
4 both
sequence a of
1
Gene trans!ation_start: compound 4
the SNP
translation_start:
2 in in parents
may human
the the
ENSG000001 1079 was
JH8. SEQ coordinates be
also heterozygote.
genome. of inferred (c.1079delC,
This
ID
the present
392 listing
results 257 68509
JH12
from The are
in in
2010202722 29 Jun 2010 20 10 15 30 25 5
found is LOC1 predicted NP_660320.1. exons HFE2A 5 For promoter ’
of similar
SEQ
the
48738
T Proteinl Translated Translated SEQ SEQ SEQ in have terminus from SEQ Human annotated SEQ paralog SEQ RGM SEQ SEQ HFE2A
SEQ ranslated Promoter
transcriptional
the ID region Ensembl to
rat ID ID ID SEQ Nos. Translated ID ID the ID ID ID ID protein
Genomic on NO. NO. NO. Protein
No. is No.
No. not NO. NO.
and
NO. Ensembl
from
as ID 19 Sequences from from the chromosome
Protein
10 12 11
26 defined 27
from 24 NOs -
exons
22
23 translation
protein 25
21, T
- - -
2 Transcript
Transcript - start -
Proteins - ranscript Proteinl
Protein DNA Protein
- is
Similar -
as
Similar Homo -
the Similar
protein Human ENSP00000304614. the
similar site. follows:
Similar
for translation fragment promoter
protein
5,
2- 3 sapiens
Human, to
ENSP00000320072 3 of
to
4 1
to ENSP00000304614 sequence
ENSP00000320072
Genomic
and gi|21758120|dbj|BAC05248.1| to
AK098165.1. SINFRUP000001 to ENSESTP00000023393
59 translation gi|243081
sequence Transcript including
ENSMUSESTP00000016634 of but Mouse
Transcript
nucleotide starting DNA
SEQ
and 89
HFE2A
of is 2 Protein fragment 1ref|NP_064
deemed
Transcript from
Rat ID
38308
1
and
sequence NOs and
promoter first 3
the
Transcript including Fugu to is are
methionine Human
596.1 2 begin similar NCBI
as
and
identified
Rat HFE2A, region
follows: 1
1500
may
protein HFE2A Human
HFE2A HFE2A Mouse to 2
and and
the
be bp as N-
2010202722 29 Jun 2010 5
the compositions
claims SEQ Those RGM
further
ID
skilled and
No.
set methods
in
28 out
the
- below.
art Similar disclosed
will
be to
above, familiar gi|22651834|gb|AAM95449.1| 60
all
with
of
which numerous
are
encompassed variations
Chicken
of
the
by
61
CLAIMS: 2012
1. An isolated antisense molecule complementary to SEQ ID NO: 2 or 3, or wherein said antisense molecule is the complement of the oligonucleotide of SEQ ID NO: Nov 13, 15, 17, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, 51 or 53, wherein said antisense 22 5 molecule modulates iron transport across a cell membrane of a cell that transports iron.
2. The isolated antisense molecule of claim 1, wherein the antisense molecule is the complement of the oligonucleotide of SEQ ID NO: 13, 15, 17, 29, 31, 33, 37, 39, 41,43,45,47, 49,51 or 53.
3. The isolated antisense molecule of claim 1, wherein said antisense io molecule is an RNA. 2010202722
4. A method for inhibiting hemojuvelin activity in a cell, comprising contacting a cell with an isolated antisense molecule of claim 1, wherein said antisense molecule inhibits hemojuvelin activity in said cell.
5. The method of claim 4, wherein said antisense molecule is the complement is of the oligonucleotide of SEQ ID NO: 13, 15, 17,29, 31,33, 37, 39, 41, 43, 45, 47, 49, 51 or 53.
6. The method of claim 4, wherein said antisense molecule is an RNA.
7. The method of claim 4, wherein said cell is a member selected from the group consisting of a macrophage, inflammatory cell, hepatocyte, CHO cell, intestinal 20 cell, hematopoietic cell, pancreatic cell, skeletal muscle cell, a cell of the nervous system, or a Caco2 cell.
8. A method for modulating iron transport across the membrane of a cell, comprising contacting a cell with an isolated antisense molecule of claim 1, wherein said antisense molecule modulates iron transport across said cell.
25 9. The method of claim 8, wherein said antisense molecule is the complement of the oligonucleotide of SEQ ID NO: 13, 15, 17, 29, 31, 33, 37, 39, 41,43, 45, 47, 49, 51 or 53.
10. The method of claim 8, wherein said antisense molecule is an RNA.
11. The method of claim 1, wherein said modulation of iron transport across a 30 cell membrane results in release of iron by said cell.
6884588 62
12. The method of claim 1, wherein said modulation of iron transport across a 2012
cell membrane results in iron uptake by said cell.
13. The method of claim 8, wherein said cell is a member selected from the Nov group consisting of a macrophage, inflammatory cell, hepatocyte, CHO cell, intestinal 5 cell, hematopoietic cell, pancreatic cell, skeletal muscle cell, a cell of the nervous system,
22 or a Caco2 cell.
14. A method for inhibiting hepcidin production in a cell, comprising contacting a cell with an isolated antisense molecule of claim 1, wherein said antisense molecule inhibits hepcidin production in said cell.
io 15. The method of claim 14, wherein said antisense molecule is the 2010202722 complement of the oligonucleotide of SEQ ID NO: 13, 15, 17, 29, 31, 33, 37, 39, 41, 43, 45,47,49,51 or 53.
16. The method of claim 14, wherein said antisense molecule is an RNA.
17. The method of claim 14, wherein said cell is a member selected from the is group consisting of a macrophage, inflammatory cell, hepatocyte, CHO cell, intestinal cell, hematopoietic cell, pancreatic cell, skeletal muscle cell, a cell of the nervous system or a Caco2 cell.
18. An isolated polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9, wherein said polypeptide modulates iron transport across a 20 cell membrane of a cell that transports iron.
19. An isolated fragment of SEQ ID NO: 26 or 27, wherein said polypeptide modulates iron transport across a cell membrane of a cell that transports iron.
20. A composition when used for treating a disease of iron metabolism comprising a therapeutically effective amount of a polypeptide of claim 18 or 19 in a
25 pharmaceutically acceptable carrier.
21. The composition of claim 20, wherein said disease is juvenile hemochromatosis, adult onset hemochromatosis, anemia of chronic disease (anemia of inflammation) or iron deficiency anemia.
Dated 22 November 2012 3o Xenon Pharmaceuticals Inc.
Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON
6884588 010202722 29 Jun 2010 1/17 2010
1 10 29 30 40 60 Μ 70 BO SO 100 HO 120 130
hunen TCTBTBTnCRTnnflflTRfWTCrflGRS«iflCnGflflRGCfiGnCTGSraiTGaCRETCTRGnTCm:TflGSTBCHTRCHCHTGi3nRTflGflTRTRCRTCTCTATnTHCftTfiCflGGTRtmTRCfl6RTnTRCnT
Jun Hus
Ret Consensus ......
131 140 ISO 160 170 180 190 200* 210 220 230 240 250 260 29
hunen RTftTGCCCTRnR6TTCTGnCCTCTRGRCRRCCCTfiH7flCflin6RCCGTflTTTGGRRTCCGTCCrTCTCTTflflrnCBCnGGCRB6TBCTRflRRGflTGflTCflTCTCRGflTfiTRCCTflT66CT6CRRRfl Hus Ret
Consensus
261 270 280 290 300 310 320 330 340 350 360 370 380 390
hunen RCftTGRMTGGCTRRRTCCCnGGTTGCR6TBrcirrrrrcrrrrnRflGGe»GGTBGBGGG6CGSGTCTCRCTGTTGCCCRGGCTG5flGTGCflRTGGCGnRlCflTe6CTCftCTGCfl6ttTCRRnCTCC Rue .Ret Consensu®
391 400 410 420 430 440 450 450 470 480 430 500 510 520
i6tXCTCBRGTORXCnXT6CCTCRGClCCCflRRGIGCTGRGRlTnGCRnTRnTRTGGTCRCRRGRnRIGnflnCanRflRR6TfllCrnciGflGGCrRGGCnTGnGGTTCRCRCnGTRRPfCC hunen Rue Ret Consensus
521 530 540 650 560 670 580 590 600 610 620 630 640 650
2010202722 CRtXnCTCTGAGfl6GCTGR0lTGGARGGAnCAnGRGGCRAGGRGnBVU3lCCRGCriGGTCflRCRTRGTGftGftCCTCSnnCGGRflGGRR&GRftG6flRGGRGG6RGIXRBaiRGGGftGGGfl6TGflRG hunen Rus Ret Consensus
651 660 670 680 699 700 710 720 730 740 750 760 770 780
6flRGfiRflGGRR6GflRGGRRGGRfl(aauttGRfl6(aWRiRftGGRRGGRflGGMRRGTflroTTnT6flRTCTTTnCTRTTTClCCRRCTCTTriCTnflGflflfiRflTTClflTTTCCRnCTnCTTCflCCTCTt hunen Hus Ret TCTSSCCCRCnCTCTGlGGRTCGGTCRTCnCRRRRCCTOnCRGCTTC-CCRCT- Consensus
781 790 800 810 820 830 840 850 860 870 880 890 900 910
hunen lGCCTnGnRGCCTTCTCTCCflftGCRflRTCGGGRCCCTn«nTTnGTGTflTTCflT6flGG6flGflGGfWGRT6RRnGCT6TfiCflRftCTRRflGTflflTGRRRflTGGRGTfiG6TRGGflGGRTR6flCRGCTG Rus TGIRTT—GRCCTGGRGGRGGGftTTGnGCflCRCflCTRGCGTRATGGRRRftGGTGGRGflTftGflR—TRGRCRGCTG Ret -GCC——CCCRCCeCCCCCGRRRCCCGGRRCCnCTGTTTnTGTGrnGTflrr—CnCCRGGRGGRGGGflTTGTTGCflCRCflCTflGCGTflATGGflflflflGGTGGR6RTRGRR—--TRGflCflGCTG Consensus •C®c« •ce,.c<. 911 820 930 940 959 860 970 980 990 1000 1010 1020 1030 1040 hunen CRRGGflTCTGRGCTCffinRGfltTGRflCflRflCCCTCflTCCTRflGCRRCICflCRGETCnGnnTCTTCTCTGGflCflGCTGGCTTTTTTCGTCCTTCTGRflflTRCTCTGCBRRGRTRBGRGflGGGGCTflrGflfl Hus CnGRGnTCTGRTCCGGBCflGntRGnBTRnflCCCTCCTCCGBflRCRmrCTGCTCTCGGGnTCTTCICCnGBCnGCTGGCCnT· —CGGC-nCTGBnflTRGTTGGCGGBGRTGGGGGBGEGTCTCTGRfl Ret CflGRGRTC7GfiTCCGGRCRG8CflGRRTRRflCCCTCCTCCGflflHCRHTTCTGCTCTC6G6nTCTTCTCCflGflCRGCTGGCCTTT—CGSC-nCTCflRRIRGnGGCG6RGRTGGGGGR(H*6lCTCTGRR Consensus CRBoGRTCTOfltCcGGflcflGflCoGnRtflRflCCCTCoTCCsflRoCRRtTCtertnCcGgTncnCICcaGRCflGCTGGCcTn..CGeC.TTCTGRRRTflgTteGCcoflSRTcGGBGRGGGtCTcTGRR 1041 .1050 10GO 1070 1080 1080 1100 1110 1120 1130 1140 1150 1160 1170 hunen CTRCCTCTttTRTGGflTCTTeTTCflffflGTCflGCTRCCTCCTflGRTRCTRTCTBTRGflnCCTflflflTGTRRTflTTCRWRTRGCflGGGRTGRRCRTGGTflflRTGflRflGGTRTC—CflRTTCCCCRCTGTRR Rus CTBCGCCTGCCRI—CCRTCTTCflRRGCCRGCTRlXTC-TRCGTRCCflTGTGTGGnflRCTCflGIGGCHTCCTCflGTRRRGK!RGGRTGnGHRC6GTGflGTG8CRGGCGTCRC8tflflflTCHCCflCC«GCC Ret CTRCGCCTGCCRT—CCflTCTTCflflRGCCRGCTRCCTC-TRCGTRCCflTGTGT6GRRRCTC86TGGCflTCCTCRGTflRRGRGRGGflTGnGRRC6GTGflGTGflC8GGCGTCflCRCflflflTCnCCRCCfiGCC Consensus CTRCp:CTGCcflT.*.cOTc7rCRflflGcCflGCTBCCTC.TRoeTBCcflTeTGTrGRReCTcfleTGBcRTccTCRGtfleflGeeeGGRIGRceflcGGT8flBrGRcRGGecTCec«CRBeT€eCCRCcegce U71 1180 1130 1200 1210 1220 1230 1240 1250 12W 1270 1280 1290 1300 hunen TTmRBRWCCRGGRGCTCRflC8TTflTTGRflHRTGCTGGRGGGCTGCCTGGRGTfiGGCRGTG2CCRC0GiIGTCflCftCRRGCTGGBflrTGGflTBTCC8flCTTGTCTGTCflTBn-TCTCTCCnXCTCCC Rus GTCTTBCRGGCCGGGflOCTCfl------TTRCTGBGflftCGCTRBRDGRC------CTG-flGT-GGCRG------GTCflTRCRflGCTOCflGTTGGflCnTGGGflCTTGGCCnTCGnCTCrCinRGGCflGTCIX Ret 6TCTTRCRGGCCGGGflQCTCfl------TTRCTGRGRRCGCIRRRGGRC------CrG-flOr-GGCflO· ■ ■ GTCaTHCBRGCreCRaTTOSHCBfGGGBCrTGCCCBICSnCTCICTrfBGOCHCTCCC Consensus BToTTRcR6GCCe6GflGCTCn,..TTRcTGRfiHRc6CTMflGGBC...CTG.fiGT.GGCflR...... GTCfltBCflflGCTGoflBTTGGflcfiTBeRflCTTGeCcaTCeTtcTcTCTtTeefiCaeTCCC 1301 1210 1320 1330 1340 1350 1360 1370 1300 1330 1400 1410 1420 1430 hunen TEflCTTGCCBCTCR8TRCTCC-flTflTTCnTCT—BflTCCTCTBfiCCCTCCCCflCTCCCCCfiRCTCCCflCflCCCTRCCCCCRCCRflCGTTCCTGDflRnTTGGBCTTflGCTRTTTTTflflRflCCGTCBBCT Hue rCRniGGTHCCtRnCanCCCBrflnCCCTCnTflTrrTGCTraTCflnCCTGCTGCtnRGCTCCCflCnaCTRCTGCIflCCflflCGnaiGGRRrnrGGniXTeGCTRnnTflflflRCTGlCRRCT Ret TniCTIGGTRCCCHGCRnCCCflTflTTCCCTCTTTBnrTGClCRTCflnCCTGCTGCCTTRGCTCCCflCRCCCTRCTGCCRCCHflCGTTCCTGGflflrTTTffiRCCTflGCTnTTTTTRRRflCTGTCflBCT Concensus TcBCTTGGtflCcCflecBtTCCcflTBTTCccTCnxfltltteCTcfltCaTtCCteCTeCCttflgCTCCCflCBCCCTBCteCCflCCBRCGTTCCTGGRRTTTTGGflCcTflGCTflTTTTTRRRflCtGTCflRCT 1431 1440 1450 1480 1470 1480 1430 1500 1510 1520 1530 1540 1550 1560 hunen CBGTRGCCRCCTCCCTCC-CTGCTCBGCTGTCCflGTRCTCTGGCCBGCCBTBTnCTC ------CCCCnnXCCCHTRCaiftnCCnCTCTGGlTCCCIGHIXICRGTGBGROnlRGCCGG-CCTGGCG Hue C8GGRGGCBCCTCCCTCCT(XTCTCflGCT6TCCfiGTGCrrGGGCC8flCCeTflTRCTCTCCCT6CCCCCTCCCCCCRCHCCflRRGCTCCTCTGGCTCTCIGRCCTCGGTGRGRTTGCflGCCflGTCCGGGGG Ret CflGGRG6CflCCTCCCTCCTCCTCTCflGCT6TCCflGTGCTTGGGCCflflCCflTflTRCTCTCCCTGCCCCCTCCCCCCnCflCCflRflGCTCCTCTGGCICTCTGRCClCGGT6flGRTTGCHGCCflGTCC6GGGG Consensus CRGiBGBCRCCTCCCTKtCctCTCRGCTGTCCnGTBCTtsGGCCflaCCnTRTRCTCUxctECCCCeTCCaCCRdlCCRRfltCTcCTCIGCclCtCTGRKICotSTRRORttGCRGCCaGtCCsGGGe 1561 1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680 1690 hunen RGCTCGGCGRGBCnCGGRG------GHCCCCCrGGCTGGflGCTGflCCCflCflGflGTRGGGnflTCRTGGCTGGBGnRTTGGBTflGCflGflGTflHTGTrninCCTCTEGfiflflCflGTflBGTCflflflGTGfl Hus nTCOGOGBCBGflCBTGGflGflRGGRGflTGGflGGBCCCCCTGGCTGGBGCBGRCCflRCnGBRTRGGCRnCTflTGGCTGGRGaRCCGGGTnrCBGflGTflHTGCnGflCCTCGGGRflRCflGTRRGrCTflGflTGI) Ret HTCG6GGflCflGflCflTGGfl6eRG6ROnGGRGGflCCCCCTGGCTGGflGCfi6RCCRRCflGflflTB66CRflCTfl7GGCTGGRGflRCCGGGTHTCB6flGTflflTGCnGHCCTC6G6RflflCHGTRflGTCTflOITGfl Consensus BtXjGGGacflGflCBtGGflOeaBBeBetBBeBGRCCCCCTGGCTGGflGCeGRCCaflCBDflaTflSGcnftctBTGGCTGGRWflccGGBTBtCRGBGTflflTOcTTGflCCTCBGGflflRCBGIRRGTCtflBBTGH 1691 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 1810 1020 hunen nflTTGCflHTTCCrrTRflTflR6CrTTTBTHn6eRGTTfiGRCTnTRTRflflflTTRCRBflCBCCTHCTTGGHTGTCTCTC6TCCflflflTGCTBGGfiTCTCTCCCT8CCRRGGT6CCCCfiflTCTCCflTTTCTCT Rus BflTGGCGGTTGCnTGBTRR6CriTT66GTCGflGGCTflGflflTTTCflTHBfl6TTRCflGRCH—~———TCTGTTCTGflflBflCTRflGBTCTCTCCTTRCCfl-OBTBCCCCflflTCT------TCBCT Ret BflTGGCC6TTGCnTGBlflBGCTTTTGCGTCBflGGCTfl6R9TTTCflTARfiGnRCR6flCR ■ TCTOnCTCaflRRCTRBGRTCTCTIXnHCCH-GaTBlXCCBHTCT------TCBCT Consensus RRTlGCeBTTeCTTTBflTBRGCTTTTBBeTcGReGcTflGflarncRTflRReTTRCflBflCR...... TCT8tTCt»BRaaCTaaGRICTCTCCtTflCCB.6aTaCCCCnRTCT...... TCaCT 1021 1830 1840 1850 1880 1870 1880 1890 1900 1910 1320 1330 1340 1946 hunen TTCIGTCTTRnTCTnCTGGCCTCTGKCTCTRGCTTTrTGRBGTTrflBTTCTCTGTCTnCCTCTGGCRGTCTTBGCCnCTCTTTRCCnBnflCCTCRBGBCTttTGRTBIBGrnTRGBflG Hus TTTGGRC------CGCCTGCTCflTRCflCnRTTCCflflnGRRG6GTTTTGflCflGGflGBRRGGGflGRCR6fiCCCCTCCCRflTBTCTQn—CC Ret rnCGRC—————CGCCTGCTCaTflCflCnenCCanRGRReGeTTTTaiCRGGRGRRRGCGRGRCRGflCCCCTCCCRaTfiTCTGTT—CC Consensus FIGURE 2 2/17 2010202722 29 Jun 2010 ~2 Η.Πω •H P'il EfliCEfi J tajtn <*> in ωτ,<2]υ 3 3 ΕίΠ +>.11 J 3EK3J 3 3 m a X> k W 0 G IK) l£H. vo J 3 J <* o 3 0 K 05 2 a g rt G IH ta; + 3 J tn co o 3 3 2 h j r* co n W & « co 04 o ° O o o o CN co ft m Hjw Η rl vo O h ♦H Ci u 0 Ui < > 2 ω σ w 3 u Ex Di Di Η < H 2 Q 3 < 2 2 a 3 •c 3 Di < xj ω CM a) a M 0 Η h cn (!) 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LL co ο I I © σ» ·· K c 3 3 E vo 00 •H Λ Λί 0 U *, *, 2 '· Φ β U €0 ci rH Λ ·· β 3 £ (rt in σ\ H •Η Λί Λ 0 ·· 0 2 υ Φ β Ci f- vo ’ a <ΰ C 3 Ε · in Cl rn -Η X Λ ·» α 0 2 υ υ φ β | 5/17 rn in o - « 3 β 3 ε m r* · Λί £3 *| 0 2 0 υ φ β η WO w o * O «· Ci © * ο © I I | | k u> Cl I I I ·· κ » 3 a a 6 •Η Λί Λ 0 2 *ι 0 υ β φ 2010202722 29 Jun 2010 I r* < λ CQ o m σι x o 3 ε « a I & w X , ε 3 β β V φ σ 1 1 - X i 0 3 4) Q oi fS m <4 4-> (0 Η in b Η tn io X 0 ε x β u υ β 1 Η u, σι •rf A Λί X ο U s 4) β υ 1 p» Η < X X n X ο 0) β 0 3 co CL 1 H © u> r4 u « Z b cn (4 C ce V 1 • dNrtHtfmnnmm Ρ·ΐηΜ·χΓθΗ<ΛΜ)10Ν r A — X W C 4) 3 ε (4 β (0 E I 1 be « « ο © π σχ 3 ε (4 β 1 <Η X QS ω 3 ε β 4) 4) ο (4 1 1 1 ε Ο 3 a V 6/17 ±> OS β b 3 C7) 3 χ ο X X β υ υ β A Ή Λ U DS ο 2 φ β υ 1 X X 0 X 4) co β Ο CL 3 1 »- U Μ ζ b β β υ σι D « I 1 rH A » X β 3 ε G Ο 4) Ε β 1 X < ΓΠ σν ’ X 0 ο 3 ε β C i* 1 1 «Η X OS ω ε β 0) 4) σ 3 β X Ο 3 Q 4) X β b 3 σι 3 0 X X X β υ β 0 1 A •Η X Ο X OS ο U φ β 1 X X X 0 4> β Ο 3 η Ο 1 rM u w <0 c v σι NP 2010202722 29 Jun 2010 7/17 2010202722 29 Jun 2010 ■c 3 c w Φ φ ra 5 S φ ’ Ο Q- > ap.Hdad leuBis ο z 8/17 < G99V I222N 1281 T G32 0V | C36 1FS X I R326X V4031 2010202722 29 Jun 2010 FIGURE FIGURE -Ω « O a .6 transmembrane 7B 7C 50 TMHMM posterior 9/17 probabilities inside for Sequenoe 2010202722 29 Jun 2010 gnl|CDD|l92 LOC148738 gnl|CDD|l92 LOC148738 FIGURE Human Chicken_RGM FIGURE 7D 7E 220 FGDPHLRTF * 3 ie 0 kb $>KVYQKg 10/17 5 240 ggkvt VD VM 9 9 * 260 48 46 2010202722 29 Jun 2010 281 421 41 341 1021 321 961 301 781 241 361 301 241 21 721 221 661 201 901 841 261 541 481 141 121 101 81 61 181 121 61 1 601 181 161 1 GATACTGCCAGACGGCTGTGCAAGGAAGGGCTTCCAGTGGAAGATGCTTACTTCCATTCC ATTGGCACAACTATAATCATTCGGCAGACAGCTGGGCAGCTCTCCTTCTCCATCAAGGTA ACCATCATATTTAAGAACATGCAGGAATGCATTGATCAGAAGGTGTATCAGGCTGAGGTG AGCACTCTCACTCTCCTGCTGCTCCTCTGTGGACATGCTCATTCTCAATGCAAGATCCTC TGCCCTCCAAGTCAGCGACTCTCTCGATCAGAGCGCAATCGTCGGGGAGCTATAACCATT GCAGAGGATGTGGCCATGGCCTTCTCAGCTGAACAGGACCTGCAGCTCTGTGTTGGGGGG GGCCTCCCTGCCCCGGACCCTTGTGACTATGAAGGCCGGTTTTCCCGGCTGCATGGTCGT CGCCAGGGCCCTACAGCCCCTCCCCCGCCCCGGGGCCCCGCCCTTCCAGGCGCGGGCTCC S^GGGAGCCAGGCCAGTCCCCTAGTCCCAGGTCCTCCCATGGCAGTCCCCCAACTCTA TCCAGTTTGTCGATTCAAACTGCTAACCCTGGGAACCATGTGGAGATCCAAGCTGCCTAC GATAATCTTCCTGTAGCCTTTGAAGATGGTTCTATCAATGGAGGTGACCGACCTGGGGGA GTCCAAGCCACCAGCTCCCCCATGGCGTTGGGGGCCAACGCTACCGCCACCCGGAAGCTC CTCGCCTTCCATTCGGCGGTACATGGCATCGAAGACCTGATGATCCAGCACAACTGCTCC CGAGCCCTCCGCTCCTATGCGCTCTGCACTCGGCGCACCGCCCGCACCTGCCGCGGGGAC GCACTTCGAGGAGGAGGAGGAGGAGGCCGGGGTGGAGGGGTGGGCTCTGGCGGCCTCTGT CACTTTCACACATGCCGTGTCCAAGGAGeTTGGCCTCTACTGGATAATGACTTCCTCTTT CCCCCGGGGTTCTTGCATTGCGCTTCCTTCGGGGACCCCCATGTGCGCAGCTTCCACCAT cgctgcaatgctgagtacgtatcgtccactctgagccttagaggtgggggtt -D--N--L--P--V--A--F--E--D--G--N--G--G--D--R--P--G--G- -R--A--L--R--S--Y--A--L-'-C--T--R--R--T--A--R--T--C--R--G--D- -R--C--N--A--E--Y--V--S--S--T--L--S--L--R--G--G--G--S--S--G- -D--T--A--R--R--L--C--K--E--G--L--P--V--E--D--A--Y--F--H--S- -C--P--P--S--Q--R--L--S--R--S--E--R--N--R--R*»G--A--1--T--I- -A--E--D--V--A--M--A--F--S--A--E--Q--D--L--Q--L--C--V--G--G- -I--G- -S--S--L--S--I--Q--T--A--N--P--G--N- -T--F--K--N--M--Q--E--C--I--D--Q--K--V--Y--Q--A--E--V- _V--Q- -H--F--H--T--C--R--V--Q--G--A--W--P--L--Ii--D--N--D--F--L--F- -P--P--G--F--L--H--C--A--S--F--G--D-*-P -G--L--P--A--P--D--P--C--D--Y--E--G--R--F--S--R--L--H--G--R- -R--Q--G--P--T--A--P--P--P--P--R--G--P--A--L--P--G--A--G--S- -L--A--F--H--S--A--V--H--G--I--E--D--L- -A--L--R--G--G--G--G--G--G--R--G--G--G--V--G--S--G--G--L--C- -M--G--E--P--G--Q--S--P--S--P--R--S--S--H--G--S--P--P--T--L- -S--T--L--T--L--L--L--L--L--C--G--H--A--H--S--Q--C--K--I--L- - -Τ--Τ- a - -T--S--S--P--M--A--L--G--A--N--A--T--A--T--R--K--L- -I--I--I 3 - -R--Q--T--A--G--Q--L--S--F--S--I--K--V- FIGURE 3 11/17 3 8A -Η- 3 - -Μ- -V- -H--V--R--S -I--Q--H--N--C--S- -Ε- -I--Q--A--A--Y- 3 - -F--H--H- C atcagga S TGTGTCTTTGATGTTTTAATTTCTGGTGATCCCAACTTTACCGTGGCAGCTCAGGCAGCA GCTGGGGTTCCTCTTTCCTCAGCAACCCTCTTAGCTCCACTCCTTTCTGGGCTCTTTGTT CTGGAGGATGCCCGAGCCTTCCTGCC CTGTGGCTTTGCATTCAGTAAGGGGACCATCAGTCCCATTACTAGTTTGGAAATGATTTG -C--V--F--D--V--L--X--Si--G--D--P~-N--F--T--V--A--A--Q--A--A- -L--W--L--C--I--Q--* -A--G--V--P--L--S--S--A--T--L--L--A A--R--A — F--L--P--D--L--E--K--L ...... FIGURE AGACTTAGAGAAGCTGCATCTCTTCCCCTCAGAT 12/17 8B ♦ — ♦ P--L--L--S--G--L--F--V- ♦ — H--L--F--P--S--D- ( ...... JX 2010202722 29 Jun 2010 σ> on ω Ο D ΰΖ Ό 5 • Ό 5 £ Ο ο OT & CZ3 <Λ 4) Η ο Ο 13/17 < Ο LL1 σ> ZD ϋ on Ο r — ( ο 04 2010202722 29 Jun 2010 14/17 o m CN cn o 2010202722 29 Jun 2010 15/17 2010202722 29 Jun 2010 Ο ■σ φ 16/17 2010202722 29 Jun 2010 σ> UJ Q ZD u_ 17/17 CN o CN CN O o o 35 O Φ <9 fc: 2010202722 29 Jun 2010 <150> <120> <110> <151> <150> <151> <150> <141> <140> <130> gccagtcccc ggaggacccc <151> <150> <151> ccaaatttct agcagagtaa cttctctggt <212> <211> <210> <170> <160> tcctgctgct tcatcagctg <400> <213> <213> <212> <211> <210> <400> <213> <212> <211> <210> Xenon N/A Juvenile and 2004-04-08 Homo DNA 2003-04-15 2003-04-09 Homo DNA Homo DNA Patentln 2003-08-28 60/498,458 2003-07-18 60/488,607 60/462,867 60/461,615 836-115PCT 62 37 2 204 149 1 3 2 1 Uses ggaagaaccg ctggctggag tccctgacct tttttcagtc tgtttgacct cctctgtgga tagtcccagg sapiens sapiens sapiens Genetics Hemochromatosis Thereof version Inc gcgcctggga acttacaggg ctggaaaca ctgacccaca cagtgagaca catg tcctcccatg 3.0 . SEQUENCE Gene gagtagggaa gcagccggcc gcagtccccc aacctggctg cttccggtca ι (HFE2A) LISTING , gataggtatg tcatggctgg tggggacctg aactctaagc aaattcacta Expression ggggagacac ggggagccag ggtaggaggg agaattggat actctcactc Products 204 180 149 120 120 60 60 2 2010 <400> 3 Jun ctcattctca atgcaagatc ctccgctgca atgctga 37 <210> 4 29 <211> 560 <212> DNA <213> Homo sapiens <400> 4 ctcattctca atgcaagatc ctccgctgca atgctgagta cgtatcgtcc actctgagcc 60 ttagaggtgg gggttcatca ggagcacttc gaggaggagg aggaggaggc cggggtggag 120 gggtgggctc tggcggcctc tgtcgagccc tccgctccta tgcgctctgc actcggcgca 180 ccgcccgcac ctgccgcggg gacctcgcct tccattcggc ggtacatggc atcgaagacc 240 tgatgatcca gcacaactgc tcccgccagg gccctacagc ccctcccccg ccccggggcc 300 ccgcccttcc aggcgcgggc tccggcctcc ctgccccgga cccttgtgac tatgaaggcc 360 2010202722 ggttttcccg gctgcatggt cgtcccccgg ggttcttgca ttgcgcttcc ttcggggacc 420 cccatgtgcg cagcttccac catcactttc acacatgccg tgtccaagga gcttggcctc 480 tactggataa tgacttcctc tttgtccaag ccaccagctc ccccatggcg ttgggggcca 540 acgctaccgc cacccggaag 560 <210> 5 <211> 1230 <212> DNA <213 > Homo sapiens • <400> 5 ctcaccatca tatttaagaa catgcaggaa tgcattgatc agaaggtgta tcaggctgag 60 gtggataatc ttcctgtagc ctttgaagat ggttctatca atggaggtga ccgacctggg 120 ggatccagtt tgtcgattca aactgctaac cctgggaacc atgtggagat ccaagctgcc 180 tacattggca caactataat cattcggcag acagctgggc agctctcctt ctccatcaag 240 gtagcagagg atgtggccat ggccttctca gctgaacagg acctgcagct ctgtgttggg 300 gggtgccctc caagtcagcg actctctcga tcagagcgca atcgtcgggg agctataacc 360 attgatactg ccagacggct gtgcaaggaa gggcttccag tggaagatgc ttacttccat 420 tcctgtgtct ttgatgtttt aatttctggt gatcccaact ttaccgtggc agctcaggca 480 gcactggagg atgcccgagc cttcctgcca gacttagaga agctgcatct cttcccctca 540 gatgctgggg ttcctctttc ctcagcaacc ctcttagctc cactcctttc tgggctcttt 600 gttctgtggc tttgcattca gtaaggggac catcagtccc attactagtt tggaaatgat 660 ttggagatac agattggcat agaagaatgt aaagaatcat taaaggaagc agggcctagg 720 agacacgtga aacaatgaca ttatccagag tcagatgagg ctgcagtcca gggttgaaat 780 tatcacagaa taaggattct gggcaaggtt actgcattcc ggatctctgt ggggctcttc 840 accaattttt ccagcctcat ttatagtaaa caaattgttc taatccattt actgcagatt 900 tcacccttat aagtttagag gtcatgaagg ttttaatgat cagtaaagat ttaagggttg 960 agatttttaa gaggcaagag ctgaaagcag aagacatgat cattagccat aagaaactca 1020 aaggaggaag acataattag ggaaagaagt ctatttgatg aatatgtgtg tgtaaggtat 1080 gttctgcttt cttgattcaa aaatgaagca ggcattgtct agctcttagg tgaagggagt 1140 ctctgctttt gaagaatggc acaggtagga cagaagtatc atccctaccc cctaactaat 1200 ctgttattaa agctacaaat tcttcacacc 1230 <210> 6 <211> 1379 <212> DNA <213> Homo sapiens <400> 6 2010202722 29 Jun 2010 ggcttccagt gttctatcaa ggaggacccc gcattgtcta gcattgatca gggagtctct gggttgagat gttggggggt ggaggacccc agacatgatc acttagagaa atcccaactt cagagcgcaa ctgaacagga cagctgggca ctgggaacca agcagagtaa cttctctggt actaatctgt aggtatgttc aactcaaagg cagatttcac ctcttcacca tgaaattatc cctaggagac aatgatttgg ctctttgttc ccctcagatg caggcagcac ataaccattg gctgcctaca gctgaggtgg agcagagtaa agaagtatca tatttgatga tttaatgatc aaattgttct ctgcattccg cagatgaggc aagaatcatt atcagtccca tcttagctcc ttccattcct atcaaggtag cctgggggat tgctgactca cttctctggt <400> <213> <212> <210? <211> Homo DNA 7 7 1416 gtgtctttga gccctccaag gctcttaggt gatctctgtg gctgcatctc ggaagatgct gctctccttc gaaggtgtat gcttttgaag tccctgacct cagaggatgt ccatcatatt ctggctggag atatgtgtgt attagccata agtaaagatt aatccattta actcctttct taccgtggca tcgtcgggga cctgcagctc tggaggtgac ctggctggag aggaagacat atttttccag acagaataag acgtgaaaca agatacagat tgtggctttg ctggggttcc tggaggatgc atactgccag ttggcacaac ccagtttgtc ataatcttcc tgtttgacct tccctgacct tccctacccc tgcagtccag aaaggaagca ttactagttt tgtggagatc tgtttgacct tattaaagct tgctttcttg ttttaagagg ccttataagt sapiens acaaattctt gtaaggtatg aatggcacag aattagggaa caagagctga cattcagtaa gaagggagtc gggctcttca gggcctagga ggaaatgatt gggctctttg gctcaggcag gctataacca ggccatggcc taagaacatg attcaaaaat ttagaggtca gattctgggc ccgagccttc ctgacccaca agaaactcaa ggttgaaatt ttcccctcag cgacctgggg cagtgagaca cctcatttat atgacattat tggcatagaa tctttcctca acggctgtgc tcagcgactc ctaactaatc taagggttga tgtgttgggg tccatcaagg caggctgagg ctgacccaca cagtgagaca gattcaaact tataatcatt tgtagccttt ctggaaacac ctgcagattt tacttccatt caagctgcct ctggaaacac tgttttaatt cacacc tctgcttttg ttctgctttc aggaggaaga gatttttaag cacccttata ccaatttttc atcacagaat gacacgtgaa tggagataca ttctgtggct atgctggggt cactggagga cctgtgtctt ttgatactgc ggtgccctcc tagcagagga acattggcac gatccagttt tggataatct tcaccatcat gagtagggaa gcagccggcc gtaggacaga gaagcaggca agaagtctat aagcagaaga tgaaggtttt agtaaacaaa aaggttactg ccagagtcag gaatgtaaag ggggaccatc gcaaccctct ctgccagact tctggtgatc aaggaagggc tctcgatcag ttctcagctg cggcagacag gctaaccctg gaagatggtt caggaatgca tcattctcaa gagtagggaa gcagccggcc tgttattaaa 3 tcatggctgg aagaatggca agtttagagg gattggcata aagtcagcga tgtggccatg gtcgattcaa tggggacctg gctacaaatt agtcccatta agcgcaatcg cataattagg aggcaagagc aaggattctg tgatgtttta cagacggctg aactataatc tcctgtagcc agtatcatcc ttgatgaata atgaggctgc aatcattaaa ttccagtgga tcatggctgg ggaaccatgt ttgatcagaa tggggacctg ttgttctaat ctatcaatgg ttgattcaaa cagcctcatt ttgcattcag tcctctttcc tgcccgagcc atttaagaac catgatcatt aatgatcagt tagctccact tagagaagct tgcaagatcc ccaactttac aacaggacct ttgtctagct cattccggat ctgggcagct acaatgacat gcatctcttc ggaagcaggg ggagatccaa ggtgtatcag aatgaagcag ggcaaggtta gaagaatgta ttcctgccag gccttctcag ctacccccta cttaggtgaa tgtgtgtgta agccataaga ccatttactg ctctgtgggg cctttctggg gcagctctgt ggggagacac caggtaggac atttctggtg tgcaaggaag aaagatttaa ctagtttgga aggtgaccga gaaagaagtc attcggcaga tttgaagatg ggggagacac tgaaagcaga tcatgaaggt tatagtaaac taaggggacc tcagcaaccc ctctctcgat actgctaacc atgcaggaat agtccagggt agatgcttac tcggggagct ctccttctcc tccgctgcaa agaattggat cgtggcagct tatccagagt agaattggat cttcacacc . 1260 1200 1140 1080 1020 1320 1379 1416 1380 1320 1260 1200 1140 1080 1020 420 480 360 300 240 900 720 660 600 540 180 120 960 840 780 780 540 480 420 300 180 360 240 960 900 840 720 660 600 120 60 60 2010202722 29 Jun 2010 ggaggacccc acaatgacat gattggcata aactataatc gttcttgcat aggaggagga tgctgagtac agcagagtaa cttctctggt tgggggttca tcaatgcaag cagtccccca acctggctgg ggaggacccc gctacaaatt cataattagg aggcaagagc agtttagagg cagcctcatt aaggattctg cagacggctg aagtcagcga tgtggccatg gtcgattcaa atttaagaac caccagctcc cacatgccgt tgccccggac ccctacagcc ccattcggcg ccgctcctat <400> <213> <212> <211> <210> ttccggtcaa agcagagtaa cttctctggt aagaatggca ttgattcaaa ttgcattcag tcctctttcc tgcccgagcc tgatgtttta tcctgtagcc <213> <212> <21'1> <210> <400> Homo DNA DNA 2143 1939 Homo 9 8 9 8 gccttctcag gtacatggca gcgctctgca gaaagaagtc ggcaaggtta gaagaatgta gtccaaggag ggaggaggcc gtatcgtcca tgaaagcaga tcatgaaggt tatagtaaac tcagcaaccc atttctggtg ctctctcgat attcggcaga actgctaacc atgcaggaat cccatggcgt tgcgcttcct ccttgtgact cctcccccgc tgtttgacct ctggctggag tccctgacct cttcacacc caggtaggac aatgaagcag tatccagagt taaggggacc ttcctgccag tgcaaggaag tttgaagatg sapiens aattcactag ataggtatgg actctaagca ctggctggag tgtttgacct tccctgacct sapiens atcctccgct tcaggagcac gggagccagg cagtgagaca ttcgaggagg gcaatgctga gtaggagggt ctctcactct ctgacccaca ctggaaacac gcattgtcta ggcttccagt gttctatcaa ctgcattccg gcattgatca ggggtggagg atcagtccca acttagagaa cagctgggca atgaaggccg cagtgagaca agacatgatc cagagcgcaa tgggggccaa atcccaactt agaagtatca aaattgttct tcttagctcc ctgaacagga cttggcctct ctcggcgcac ctgacccaca tatttgatga cagatgaggc aagaatcatt ctgggaacca tttaatgatc tcggggaccc cccggggccc tcgaagacct ctctgagcct ctggaaacac 1 gtacgtatcg gagtagggaa gatctctgtg gctctccttc gttttcccgg gatgatccag catcagctgg gcagccggcc aatccattta tgcagtccag tgtggagatc gaaggtgtat ggtgggctct gagtagggaa gcagccggcc gctcttaggt gctgcatctc ggaagatgct tcgtcgggga aggaggagga cctgctgctc tccctacccc tggaggtgac actggataat cgcccttcca cgcccgcacc tagaggtggg tcattctcaa ccagtcccct atatgtgtgt attagccata agtaaagatt taccgtggca caaatttctt aaaggaagca cgctaccgcc ccatgtgcgc ttactagttt actcctttct cctgcagctc 4 gaagaaccgg gtaaggtatg gggcctagga ggccggggtg ggttcatcag gaagggagtc gggctcttca gggctctttg gctcaggcag gctataacca cgacctgggg caggctgagg ggcgcgggct ttttcagtca tggggacctg agaaactcaa tccatcaagg gacttcctct tcatggctgg ggttgaaatt ggaaatgatt ggcggcctct tccactctga ctctgtggac agtcccaggt tcatggctgg ctaactaatc taagggttga ttcccctcag tgtgttgggg acccggaagc agcttccacc tgccgcgggg tacttccatt caagctgcct ctgcatggtc cacaactgct tgcaagatcc tggggacctg ctgcagattt gtcccccggg gagcacttcg gatttttaag gaggggtggg tctgcttttg gacacgtgaa cactggagga tagcagagga gatccagttt atcactttca cccgccaggg ggggagacac gccttagagg cctcccatgg cgcctgggaa ggggagacac aggaggaaga cacccttata tggagataca ggtgccctcc gtcgagccct agaattggat acattggcac ccggcctccc tccgctgcaa agaattggat tgttattaaa ccaatttttc atcacagaat atgctggggt cctgtgtctt acctcgcctt atgctcattc cttacagggc tggataatct tcaccatcat ttgtccaagc ttctgctttc ttctgtggct ttgatactgc 1080 1020 1380 1320 1260 1200 1620 1560 1140 1920 1939 1860 1800 1740 1680 1440 1500 420 480 360 300 240 120 540 180 960 780 720 660 600 840 900 240 480 420 360 300 180 120 60 60 5 2010 ctctggcggc ctctgtcgag ccctccgctc ctatgcgctc tgcactcggc gcaccgcccg 540 Jun cacctgccgc ggggacctcg ccttccattc ggcggtacat ggcatcgaag acctgatgat 600 ccagcacaac tgctcccgcc agggccctac agcccctccc ccgccccggg gccccgccct 660 tccaggcgcg ggctccggcc tccctgcccc ggacccttgt gactatgaag gccggttttc 29 720 ccggctgcat ggtcgtcccc cggggttctt gcattgcgct tccttcgggg acccccatgt 780 gcgcagcttc caccatcact ttcacacatg ccgtgtccaa ggagcttggc ctctactgga 840 taatgacttc ctctttgtcc aagccaccag ctcccccatg gcgttggggg ccaacgctac 900 cgccacccgg aagctcacca tcatatttaa gaacatgcag gaatgcattg atcagaaggt 960 gtatcaggct gaggtggata atcttcctgt agcctttgaa gatggttcta tcaatggagg 1020 tgaccgacct gggggatcca gtttgtcgat tcaaactgct aaccctggga accatgtgga 1080 gatccaagct gcctacattg gcacaactat aatcattcgg cagacagctg ggcagctctc 1140 cttctccatc aaggtagcag aggatgtggc catggccttc tcagctgaac aggacctgca 1200 gctctgtgtt ggggggtgcc ctccaagtca gcgactctct cgatcagagc gcaatcgtcg 1260 gggagctata accattgata ctgccagacg gctgtgcaag gaagggcttc cagtggaaga 1320 tgcttacttc cattcctgtg tctttgatgt tttaatttct ggtgatccca actttaccgt 1380 2010202722 ggcagctcag gcagcactgg aggatgcccg agccttcctg ccagacttag agaagctgca 1440 tctcttcccc tcagatgctg gggttcctct ttcctcagca accctcttag ctccactcct 1500 ttctgggctc tttgttctgt ggctttgcat tcagtaaggg gaccatcagt cccattacta 1560 gtttggaaat gatttggaga tacagattgg catagaagaa tgtaaagaat cattaaagga 1620 agcagggcct aggagacacg tgaaacaatg acattatcca gagtcagatg aggctgcagt 1680 ccagggttga aattatcaca gaataaggat tctgggcaag gttactgcat tccggatctc 1740 tgtggggctc ttcaccaatt tttccagcct catttatagt aaacaaattg ttctaatcca 1800 tttactgcag atttcaccct tataagttta gaggtcatga aggttttaat gatcagtaaa 1860 gatttaaggg ttgagatttt taagaggcaa gagctgaaag cagaagacat gatcattagc 1920 cataagaaac tcaaaggagg aagacataat tagggaaaga agtctatttg atgaatatgt 1980 gtgtgtaagg tatgttctgc tttcttgatt caaaaatgaa gcaggcattg tctagctctt 2040 aggtgaaggg agtctctgct tttgaagaat ggcacaggta ggacagaagt atcatcccta 2100 ccccctaact aatctgttat taaagctaca aattcttcac acc 2143 <210> ' 10 <211> 200 <212> PRT <213> Homo sapiens <400> 10 Met Gin Glu Cys He Asp Gin Lys Val Tyr Gin Ala Glu Val Asp Asn 1 5 10 15 Leu Pro Val Ala Phe Glu Asp Gly Ser lie Asn Gly Gly Asp Arg Pro 20 25 30 Gly Gly Ser Ser Leu Ser lie Gin Thr Ala Asn Pro Gly Asn His Val 35 40 45 Glu lie Gin Ala Ala Tyr He Gly Thr Thr lie He He Arg Gin Thr 50 55 60 Ala Gly Gin Leu Ser Phe Ser He Lys Val Ala Glu Asp Val Ala Met 65 70 75 80 Ala Phe Ser Ala Glu Gin Asp Leu Gin Leu Cys Val Gly Gly Cys Pro 85 90 95 Pro Ser Gin Arg Leu Ser Arg Ser Glu Arg Asn Arg Arg Gly Ala He 100 105 110 6 2010 Jun Thr He Asp Thr Ala Arg Arg Leu Cys Lys Glu Gly Leu Pro Val Glu 115 120 125 29 Asp Ala Tyr Phe His Ser Cys Val Phe Asp Val Leu He Ser Gly Asp 130 135 140 Pro Asn Phe Thr Val Ala Ala Gin Ala Ala Leu Glu Asp Ala Arg Ala 145 150 155 160 Phe Leu Pro Asp Leu Glu Lys Leu His Leu Phe Pro Ser Asp Ala Gly 165 170 175 Val Pro Leu Ser Ser Ala Thr Leu Leu Ala Pro Leu Leu Ser Gly Leu 180 185 190 2010202722 Phe Val Leu Trp Leu Cys lie Gin 195 200 <210> 11 <211> 313 <212> PRT <213> Homo sapiens <400> 11 Met He Gin His Asn Cys Ser Arg Gin Gly Pro Thr Ala Pro Pro Pro 1 S 10 15 Pro Arg Gly Pro Ala Leu Pro Gly Ala Gly Ser Gly Leu Pro Ala Pro 20 25 30 Asp Pro Cys Asp Tyr Glu Gly Arg Phe Ser Arg Leu His Gly Arg Pro 35 40 45 Pro Gly Phe Leu His Cys Ala Ser Phe Gly Asp Pro His Val Arg Ser 50 55 60 Phe His His His Phe His Thr Cys Arg Val Gin Gly Ala Trp Pro Leu 65 70 75 80 Leu Asp Asn Asp Phe Leu Phe Val Gin Ala Thr Ser Ser Pro Met Ala 85 90 95 Leu Gly Ala Asn Ala Thr Ala Thr Arg Lys Leu Thr lie lie Phe Lys 100 105 110 Asn Met Gin Glu Cys He Asp Gin Lys Val Tyr Gin Ala Glu Val Asp 115 12 0 125 Asn Leu Pro Val Ala Phe Glu Asp Gly Ser He Asn Gly Gly Asp Arg 130 135 140 Pro Gly Gly Ser Ser Leu Ser He Qin Thr Ala Asn Pro Gly Asn His 145 150 155 160 2010202722 29 Jun 2010 Ala Arg Asp Cys Gly Ala Gly Met Thr Val Met Glu Ser Pro Leu 225 Pro 65 <211> <2L0> 305 He 1 <400> <213> <212> Ala Ala Arg Ala Val Thr Glu Gly Thr Gly Phe Asp His 290 Pro 210 Pro Pro Phe 50 Homo ■ 12 PRT 426 12 Leu Asn Ala Gly Thr Glu Val Gly Gly Pro Ser Phe Leu Leu Ser 275 He lie 35 195 Asp Gin Arg Asp Gin Gly Gin Ser Tyr Gin Leu Leu Leu Pro 260 100 Ser 20 Pro Phe 180 sapiens Arg Asp Thr Ala Ala Thr Leu Gly Trp Leu Leu Ser Ser 245 Phe 165 Gly Cys 5 Ser 85 Ala Arg Ala Glu Leu Val His Leu Ser Ala Arg Thr Gin Leu Tyr Lys 310 Ser 230 70 Ala Ala Arg Gin Tyr Ala Gly Glu Phe 215 Gly Leu Ser Ser Phe 55 Ser Cys 295 lie ( Arg Asp Arg Ala His Thr Lys Ser Thr Pro 280 Cys 200 lie Leu Gly Gly Leu 40 He Val Leu Gly Leu Gin Leu Ser Gly Arg Leu Gin Leu 265 lie Ser Gly Ser 185 Cys 25 105 His Ala Gin Lys Thr Cys Glu 170 Ala Val Leu Phe Thr Leu Leu 250 Ser Cys Pro 015 10 90 7 Val Arg Thr Ala Asp Lys Leu Ala Leu Val Arg 235 Arg Asn Gly Ser 75 Ala Asn Val Glu Cys Leu 220 He Phe Ala His Arg Gly Leu Ser Pro Ser 300 60 Arg Val Glu Leu Gly Pro Glu 205 lie Ala Leu 285 Gly Gly Thr Glu Leu Ser 45 Asp Arg Gly Asp lie Leu 190 270 Ser His Leu Gly lie Ala Leu Cys Tyr He 30 110 Val Arg Gly Asp Ala Gly 255 Ser Pro 175 Arg Gly Glu Arg Val Gly Ser Leu 95 Ala Gin Ala Ala Arg Gly Val Cys Gly 240 Asp His Gly Thr Cys Ser Ser 80 8 2010 His Jun Leu Met He Gin Asn Cys Ser Arg Gin Gly Pro Thr Ala Pro Pro 115 120 125 29 Pro Pro Arg Gly Pro Ala Leu Pro Gly Ala Gly Ser Gly Leu Pro Ala 130 135 140 Pro Asp Pro Cys Asp Tyr Glu Gly Arg Phe Ser Arg Leu His Gly Arg 145 150 155 160 Pro Pro Gly Phe Leu His Cys Ala Ser Phe Gly Asp Pro His Val Arg 165 170 175 Ser Phe His His His Phe His Thr Cys Arg Val Gin Gly Ala Trp Pro 180 185 190 2010202722 Leu Leu Asp Asn Asp Phe Leu Phe Val Gin Ala Thr Ser Ser Pro Met 195 200 205 Ala Leu Gly Ala Asn Ala Thr Ala Thr Arg Lys Leu Thr lie lie Phe 210 215 220 Lys Asn Met Gin Glu Cys He Asp Gin Lys Val Tyr Gin Ala Glu Val 225 230 235 240 Asp Asn Leu Pro Val Ala Phe Glu Asp Gly Ser lie Asn Gly Gly Asp 245 250 255 Arg Pro Gly Gly Ser Ser Leu Ser He Gin Thr Ala Asn Pro Gly Asn 260 265 270 His Val Glu lie Gin Ala Ala Tyr He Gly Thr Thr He He lie Arg 275 280 285 Gin Thr Ala Gly Gin Leu Ser Phe Ser He Lys Val Ala Glu Asp Val 290 295 300 Ala Met Ala Phe Ser Ala Glu Gin Asp Leu Gin Leu Cys Val Gly Gly 305 310 315 320 Cys Pro Pro Ser Gin Arg Leu Ser Arg Ser Glu Arg Asn Arg Arg Gly 325 330 335 Ala lie Thr He Asp Thr Ala Arg Arg Leu Cys Lys Glu Gly Leu Pro 340 345 350 Val Glu Asp Ala Tyr Phe His Ser Cys Val Phe Asp Val Leu He Ser 355 360 365 Gly Asp Pro Asn Phe Thr Val Ala Ala Gin Ala Ala Leu Glu Asp Ala 370 375 380 Arg Ala Phe Leu Pro Asp Leu Glu Lys Leu His Leu Phe Pro Ser Asp 385 390 395 400 Ala Gly Val Pro Leu Ser Ser Ala Thr Leu Leu Ala Pro Leu Leu Ser 9 2010 405 410 415 Jun Gly Leu Phe Val Leu Trp Leu Cys lie Gin 420 425 29 <210> 13 <211> 21 <212> DNA <213> Artificial <220> <223> Polynucleotide replication primer <400> 13 2010202722 gctgcctaca ttggcacaac t 21 <210> 14 <211> 21 <212> DNA <213> Artificial <220> <223> Polynucleotide replication primer <400> 14 gctgcctaca ctggcacaac t 21 <210> 15 <211> 21 <212> DNA <213> Artificial <220> <223> Polynucleotide replication primer <400> 15 tgtgttgggg ggtgccctcc a 21 <210> 16 <211> 21 <212> DNA <213> Artificial <220> <223> Polynucleotide replication primer <400> 16 tgtgttgggg tgtgccctcc a 21 <210> 17 <211> 21 10 2010 <212> DNA Jun <213> Artificial 29 <220> <223> Polynucleotide replication primer <400> 17 tacttccatt cctgtgtctt t 21 <210> 18 <211> 20 <212> DNA <213> Artificial 2010202722 <220> <223> Polynucleotide replication primer <400> 18 tacttccatt ctgtgtcttt 20 <210> 19 <211> 1919 <212> DNA <213> Homo sapiens <400> 19 tctatataga tattaataaa tctagagaga cagaaagcag actggtgatg gccagtctag 60 atggctagat agatagacat ggatatagat atagatctct atatagatag aggtagatac 120 agatatagat atatgcccta ttagttctgt tcctctagag aaccctaata cagtgaccgt 180 atttggaatc ggtccttctg ttaatttcac ttggcaagta ctaaaagatg atgatctcag 240 atatacctat ggctgcaaaa acatgacatg gctaaatccc ttggttgcag tatctctttt 300 cttttttaag gggggtgggg gggcgggtct cactgttgcc caggctggag tgcaatggcg 360 ttatcatagc tcactgcagc ctcaaactcc tgcgctcaag tgaccctcct gcctcagctc 420 ccaaagtgct gagattttgc aatatttatg gtcacaagat tatgttattc cataaaagta 480 tctttctgag gctaggcatg ttggttcaca cttgtaatcc cagcactctg agaggctgag 540 atggaaggat'tcattgaggc aaggagttca agaccagcct ggtcaacata gtgagacctc 600 atctcggaag gaaggaagga aggagggagg gaggaaggga gggagtgaag gaaggaagg'a 660 aggaaggaag gaaggaagga aggaaggaag gaaggaagga aaagtatatt tttgaatctt 720 tttctatttc tccaactctt tctttagaag aattctattt ccattctttc ttcacctctt 780 tgcctttgtt agccttctct ccaagcaaat cgggagcctt tattttttgt gtattcatga 840 gggagaggaa gatgaattgc tgtacaaact aaagtaatga aaatggagta ggtaggagga 900 tagacagctg caaggatctg agctggatag actgaacaaa ccctcatcct aagcaactca 960 cagctcagat ttcttctctg gacagctggc ttttttcgtc cttctgaaat actctgcaaa 1020 gataggagag gggctatgaa ctacctctgc tatggatctt attcaaagtc agctacctcc 1080 tagatactat ctgtagaacc taaatgtaat attcagcata gcagggatga acatggtaaa 1140 tgaaaggtat ccaattgccc actgtaattt ttaaaggcca ggagctcaac attattgaaa 1200 atgctggagg gctgcctgga gtaggcagtg accacagagt cacacaagct ggaattggat 1260 atccaacttg tctgtcatat ttctctcctc cctccctgac ttggcactca atactccata 1320 ttctttctaa tcctctaacc ctccccactc ccccaactcc cacaccctac ccccaccaac 1380 gttcctggaa ttttggactt agctattttt aaaaccgtca actcagtagc cacctccctc 1440 cctgctcagc tgtccagtac tctggccagc catatactcc cccttccccc cataccaaac 1500 cttctctggt tccctgacct cagtgagaca gcagccggcc tggggacctg ggggagacac 1560 ggaggacccc ctggctggag ctgacccaca gagtagggaa tcatggctgg agaattggat 1620 agcagagtaa tgtttgacct ctggaaacag taagtcaaaa tgaaattgca attcctttaa 1680 11 2010 taagctttta tattgaagtt agacttttat aaaattacaa acacctactt ggatgtctct 1740 Jun cgtccaaatg ctgggatctc tccctaccaa ggtgccccaa tctccatttc tctttctgtc 1000 ttatttcttt ctggcctctg gcctctagct ttttgaagtt taattctctg tctctcctct 1860 ggcagtctta gccctctctt taccttatta cctcaagact cctgatgaag ttttagaag 1919 29 <210> 20 <211> 1020 <212> DNA . <213> Mub musculus <400> 20 tgtattgacg aggaggaggg attgttgcac acactagcgt aatggaaaag gtggagatag 60 aatagacagc tgcagagatc tgatccggac agacagaata aaccctcctc cgaaacaatt 120 ctgctctcgg gtttcttctc cagacagctg gcctttcggc ttctgaaata gttggcggag 180 2010202722 atgggggagg gtctctgaac tacgcctgcc atccatcttc aaagccagct acctctacgt 240 accatgtgtg gaaactcagt ggcatcctca gtaaagagag gatgagaacg gtgagtgaca 300 ggcgtcacac aaatcaccac cagccgtctt acaggccggg agctcattac tgagaacgct 360 aaaggacctg agtggcaggt catacaagct gcagttggac atgggacttg gccatcgttc 420 tctctttagg cagtccctca cttggtaccc agcattccca tattccctct ttattttgct 480 catcattcct gctgccttag ctcccacacc ctactgccac caacgttcct ggaattttgg 540 acctagctat ttttaaaact gtcaactcag gaggcacctc cctcctcctc tcagctgtcc 600 agtgcttggg ccaaccatat actctccctg ccccctcccc ccacaccaaa gctcctctgg 660 ctctctgacc tcggtgagat tgcagccagt ccgggggatc ggggacagac atggagaagg 720 agatggagga ccccctggct ggagcagacc aacagaatag gcaactatgg ctggagaacc 780 gggtatcaga gtaatgcttg acctcgggaa acagtaagtc tagatgaaat ggcggttgct 840 ttgataagct tttgggtcga ggctagaatt tcataaagtt acagacatct gttctgaaaa 900 ctaagatctc tccttaccag ataccccaat cttcactttt ggaccgcctg ctcatacact 960 tattccaaag aagggttttg acaggagaaa gggagacaga cccctcccaa tatctgttcc 1020 <210> 21 <211> 1120 <212> DNA <213> Rattus rattus <400> 21 tctggcccca cttctctgtg gatcggtcat cttcaaaacc agttcagctt cccactgccc 60 ccaccacccc cgaaacccgg aagcttctgt tttttgtgtt tgtattgacg aggaggaggg 120 attgttgcac acactagcgt aatggaaaag gtggagatag aatagacagc tgcagagatc 180 tgatccggac agacagaata aaccctcctc cgaaacaatt ctgctctcgg gtttcttctc 240 cagacagctg gcctttcggc ttctgaaata gttggcggag atgggggagg gtctctgaac 300 tacgcctgcc atccatcttc aaagccagct acctctacgt accatgtgtg gaaactcagt 360 ggcatcctca gtaaagagag gatgagaacg gtgagtgaca ggcgtcacac aaatcaccac 420 cagccgtctt acaggccggg agctcattac tgagaacgct aaaggacctg agtggcaggt 480 catacaagct gcagttggac atgggacttg gccatcgttc tctctttagg cagtccctca 540 cttggtaccc agcattccca tattccctct ttattttgct catcattcct gctgccttag 600 ctcccacacc ctactgccac caacgttcct ggaattttgg acctagctat ttttaaaact 660 gtcaactcag gaggcacctc cctcctcctc tcagctgtcc agtgcttggg ccaaccatat 720 actctccctg ccccctcccc ccacaccaaa gctcctctgg ctctctgacc tcggtgagat 780 tgcagccagt ccgggggatc ggggacagac atggagaagg agatggagga ccccctggct 840 ggagcagacc aacagaatag gcaactatgg ctggagaacc gggtatcaga gtaatgcttg 900 acctcgggaa acagtaagtc tagatgaaat ggcggttgct ttgataagct tttgggtcga 960 ggctagaatt tcataaagtt acagacatct gttctgaaaa ctaagatctc tccttaccag 1020 ataccccaat cttcactttt ggaccgcctg ctcatacact tattccaaag aagggttttg 1080 acaggagaaa gggagacaga cccctcccaatatctgttcc 1120 12 2010 Jun <210> 22 <211> 7265 29 <212> DNA <213> Homo sapiens <400> 22 tctatataga tattaataaa tctagagaga cagaaagcag actggtgatg gccagtctag 60 atggctagat agatagacat ggatatagat atagatctct atatagatag aggtagatac 120 agatatagat atatgcccta ttagttctgt tcctctagag aaccctaata cagtgaccgt 180 atttggaatc ggtccttctg ttaatttcac ttggcaagta ctaaaagatg atgatctcag 240 atatacctat ggctgcaaaa acatgacatg gctaaatccc ttggttgcag tatctctttt 300 cttttttaag gggggtgggg gggcgggtct cactgttgcc caggctggag tgcaatggcg 360 ttatcatagc tcactgcagc ctcaaactcc tgcgctcaag tgaccctcct gcctcagctc 420 2010202722 ccaaagtgct gagattttgc aatatttatg gtcacaagat tatgttattc cataaaagta 480 tctttctgag gctaggcatg ttggttcaca cttgtaatcc cagcactctg agaggctgag 540 atggaaggat tcattgaggc aaggagttca agaccagcct ggtcaacata gtgagacctc 600 atctcggaag gaaggaagga aggagggagg gaggaaggga gggagtgaag gaaggaagga 660 aggaaggaag gaaggaagga aggaaggaag gaaggaagga aaagtatatt tttgaatctt 720 tttctatttc tccaactctt tctttagaag aattctattt ccattctttc ttcacctctt 780 tgcctttgtt agccttctct ccaagcaaat cgggagcctt tattttttgt gtattcatga 840 gggagaggaa gatgaattgc tgtacaaact aaagtaatga aaatggagta ggtaggagga 900 tagacagctg caaggatctg agctggatag actgaacaaa ccctcatcct aagcaactca 960 cagctcagat ttcttctctg gacagctggc ttttttcgtc cttctgaaat actctgcaaa 1020 gataggagag gggctatgaa ctacctctgc tatggatctt attcaaagtc agctacctcc 1080 tagatactat ctgtagaacc taaatgtaat attcagcata gcagggatga acatggtaaa 1140 tgaaaggtat ccaattgccc actgtaattt ttaaaggcca ggagctcaac attattgaaa 1200 atgctggagg gctgcctgga gtaggcagtg accacagagt cacacaagct ggaattggat 1260 atccaacttg tctgtcatat ttctctcctc cctccctgac ttggcactca atactccata 1320 ttctttctaa tcctctaacc ctccccactc ccccaactcc cacaccctac ccccaccaac 1380 gttcctggaa ttttggactt agctattttt aaaaccgtca actcagtagc cacctccctc 1440 cctgctcagc tgtccagtac tctggccagc catatactcc cccttccccc cataccaaac 1500 cttctctggt tccctgacct cagtgagaca gcagccggcc tggggacctg ggggagacac 1560 ggaggacccc ctggctggag ctgacccaca gagtagggaa tcatggctgg agaattggat 1620 agcagagtaa tgtttgacct ctggaaacag taagtcaaaa tgaaattgca attcctttaa 1680 taagctttta tattgaagtt agacttttat aaaattacaa acacctactt ggatgtctct 1740 cgtccaaatg ctgggatctc tccctaccaa ggtgccccaa tctccatttc tctttctgtc 1800 ttatttcttt ctggcctctg gcctctagct ttttgaagtt taattctctg tctctcctct 1860 ggcagtctta gccctctctt taccttatta cctcaagact cctgatgaag ttttagaagg 1920 agttccctac gtcctctatt ctgtagtttt cttaccaagg ccaaatatga cctcagatga 1980 tgagtcactg atacccttct atcctgcccc cacttagcaa tgcccttcac attgagattc 2040 caagcatggg ggctgctccc tgtaaatgat ttctccccac aactctagtc cctccattct 2100 attctccctc ttgcaggact cttcccccaa tcatatcctt acccataaga taggggagtt 2160 aggcaggagg gatttagccc ctctccaact cctgtcatca taaaagactg agaacttcag 2220 aatttgaaaa gaagagatta atggaaggag tgatatttgg gaaaatacaa gaactgttga 2280 cttagaaaaa acaaatattg atttgcatgt ttggtttgca tcccattatt ccatgagaga 2340 gggagattaa aattgcagct ctctagagct gatgaaaaga gattggtttc cttttcattt 2400 gaatactgat attctagacg ggatgggtat gccaccctta atccttcttg tgttctgaca 2460 caaaggagga aaagaaatgt atgactccta gagggcatct cctcctaatg gagagggaca 2520 aataagaagt atgtttctga aatattttca ggtcctaatt ttactagggt acccactagg 2580 attactggta tctgatctag ccccatgatt cctccatctt tgacatacct gctgtttggt 2640 agctcagaat ggagcaatac agtggactct gcccccttga gttcactcaa ccttccctcc 2700 acccccacct aggggtattg cacaagggcc ctaaaagtgg ccacaggaac agggcaaaga 2760 ggcttaactg ccacacttat agtttgagga actccaatct ccccaaattc cagtctgttc 2820 atccttttct tgatctcccc agattcactc cacattatcc ttaccaatct tcaattcttc 2880 13 2010 tctctctcca tgtccagcca aatttctttt ttcagtcact tacagggctt ccggtcaaaa 2940 Jun ttcactaggt aggagggtca tcagctggga agaaccggcg cctgggaaac ctggctggat 3000 aggtatgggg gagccaggcc agtcccctag tcccaggtcc tcccatggca gtcccccaac 3060 29 tctaagcact ctcactctcc tgctgctcct ctgtggacat ggtaaggaag ggccagggaa 3120 gggtttgggg aaatctagag ggtaggctgc tatgtagggg tgggcatgtg agcctgaatg 3180 agtgaggaga gataggcgct gagagtcccg atcactcgcc ctgctctcaa atactaatat 3240 tttatttccc gttcagtctg gggaaggcca ctggggaagc ccttggtcga caggcagaag 3300 agatgtggca ggcttacaca cttttagtaa gacagccgag agaactaggg actagggggt 3360 tgggggctgg ggaaggccct tagttaggtt ttaggaaggc tggaaacccc tgatgagatt 3420 tggaagagtt atgagcaaac tacactccga tagagcagag gtctgaggac cgtctcacaa 3480 tcctctccct tctgtcttta gctcattctc aatgcaagat cctccgctgc aatgctgagt 3540 acgtatcgtc cactctgagc cttagaggtg ggggttcatc aggagcactt cgaggaggag 3600 gaggaggagg ccggggtgga ggggtgggct ctggcggcct ctgtcgagcc ctccgctcct 3660 atgcgctctg cactcggcgc accgcccgca cctgccgcgg ggacctcgcc ttccattcgg 3720 cggtacatgg catcgaagac ctgatgatcc agcacaactg ctcccgccag ggccctacag 3780 2010202722 cccctccccc gccccggggc cccgcccttc caggcgcggg ctccggcctc cctgccccgg 3840 acccttgtga ctatgaaggc cggttttccc ggctgcatgg tcgtcccccg gggttcttgc 3900 attgcgcttc cttcggggac ccccatgtgc gcagcttcca ccatcacttt cacacatgcc 3960 gtgtccaagg agcttggcct ctactggata atgacttcct ctttgtccaa gccaccagct 4020 cccccatggc gttgggggcc aacgctaccg ccacccggaa ggtcaggcac tcaatcttcc 4080 ttccgatcca cctcatgaga ttcttccacg ggcaccattc ctccccatcc ccactattca 4140 acagcaatgc tccctaattc ccttttcttc ctcaacctct cccccatctc gaatcactcc 4200 cttctaccaa acacctggag ctgtaaatca cttccccttg atgggaattt gactcaaatg 4260 cagaaaacct tgaagagaca gtcggagagg gcggacctga ggagtttcag aagggaaact 4320 tttccctctc ctaggaagtt gccacgatta agtagagagg gggttaagta gggatgaggt 4380 aatactggaa cataaatagg agaagggatc aaggattgag ggccatagta gtcctgcatc 4440 tctacttgga tcagatctct aactatgtat gaggtctgat tggggggaag atgcactgaa 4500 cccaaaatga actgttttcc ctcttgtcct cacagctcac catcatattt aagaacatgc 4560 aggaatgcat tgatcagaag gtgtatcagg ctgaggtgga taatcttcct gtagcctttg 4620 aagatggttc tatcaatgga ggtgaccgac ctgggggatc cagtttgtcg attcaaactg 4680 ctaaccctgg gaaccatgtg gagatccaag ctgcctacat tggcacaact ataatcattc 4740 ggcagacagc tgggcagctc tccttctcca tcaaggtagc agaggatgtg gccatggcct 4800 tctcagctga acaggacctg cagctctgtg ttggggggtg ccctccaagt cagcgactct 4860 ctcgatcaga gcgcaatcgt cggggagcta taaccattga tactgccaga cggctgtgca 4920 aggaagggct tccagtggaa gatgcttact tccattcctg tgtctttgat gttttaattt 4980 ctggtgatcc caactttacc gtggcagctc aggcagcact ggaggatgcc cgagccttcc 5040 tgccagactt agagaagctg catctcttcc cctcagatgc tggggttcct ctttcctcag 5100 caaccctctt agctccactc ctttctgggc tctttgttct gtggctttgc attcagtaag 5160 gggaccatca gtcccattac tagtttggaa atgatttgga gatacagatt ggcatagaag 5220 aatgtaaaga atcattaaag gaagcagggc ctaggagaca cgtgaaacaa tgacattatc 5280 cagagtcaga tgaggctgca gtccagggtt gaaattatca cagaataagg attctgggca 5340 aggttactgc attccggatc tctgtggggc tcttcaccaa tttttccagc ctcatttata 5400 gtaaacaaat tgttctaatc catttactgc agatttcacc cttataagtt tagaggtcat 5460 gaaggtttta atgatcagta aagatttaag ggttgagatt tttaagaggc aagagctgaa 5520 agcagaagac atgatcatta gccataagaa actcaaagga ggaagacata attagggaaa 5580 gaagtctatt tgatgaatat gtgtgtgtaa ggtatgttct gctttcttga ttcaaaaatg 5640 aagcaggcat tgtctagctc ttaggtgaag ggagtctctg cttttgaaga atggcacagg 5700 taggacagaa gtatcatccc taccccctaa ctaatctgtt attaaagcta caaattcttc 5760 acaccatcct ctgttgccta tgttgaatct ctttacagat gcttgaaatg gagtaaatgc 5820 aatgtgttca ctccactgaa agagggctcg gaagtatcag atactgttgc tatctcaggg 5880 agtttacagg ctattggaga gacaaaacca attcacatga aagagtgatg agtgtgtaat 5940 tattcactaa atcctacagt atggtacatt cagatgggaa gatggtagat ttgaactaaa 6000 gtaataagaa taataaaagg taacagagaa gatgggattt gaagtgagct ttgaagactg 6060 ggtaagattc aaattgttaa cgatcttcca ggcaatgaaa accatctgga gttatgcatg 6120 gattcatgat tcagcaagga aatgagcaaa actcaaatgc agtagacaag gagtaatggg 6180 acagaaggtt agatgggcag aggccagatt atgaagcacc ttaaaaaggg ggtaaggggt 6240 14 2010 ttaaatttga ttaatatcta acacttattg aaacttaaaa tctgccaggc aatgttttaa 6300 Jun acacttttaa aacattgact taattctcat agctctctaa ggaaggtggt attcttatct 6360 ctatttttat ataaggaaac ttgcctccag tcacacagct aacaaattat ggaacttgcc 6420 tccagtcaca cagttaacaa atggcagagc cagaaactga acctatgccg tttggatccc 6480 29 gaaacttaat ttttaatcac tatactatat tgtcaaggaa gcagagagcc attaaagatt 6540 ctggttgtgg agctggtaaa gattctaaaa ggggagttgt ggtggtcaat gtcaccacaa 6600 aagctactct gaggctgggc gcggtggctg acacctgtaa tccagcactt tggtaggcca 6660 atgtgggtgg atcacttgag gccaggagtt cgagaccagc ctgggcaaca tggtaaaacc 6720 ccatctctac taaaaataca agaaatttgc caggcatgat agtccatgcc tgtaatcctg 6780 taatccaagc tactcaggag gcagaggcat gagaatcgag aattgcttga acccgggcca 6840 ggaggcagag gttgcagtga gctgagacca cgccactgca ctccagcctg ggcaacagag 6900 tgagactatc aaacaaaaaa caactactct cttctcatat tctcatataa agccaaaaag 6960 agagagttgg aaaaggaggg aaagcccaaa gttgaaggaa tctagtttgt agaagaaaga 7020 ctgagaagaa atgctgttct agatgagggt gctctaaagt aacaatgctg ctctgaacaa 7080 aattatgaag gaggtaactt ttacatttaa tatcttccct gtttctggtc tcatcctccc 7140 2010202722 ctctcaggta ctccataacc gaggacctgt cctccctgcc ctaatcaagt actcaccatt 7200 atcttccacc tctcctctca gtcdctgaca cccgacaata ctcccctgaa caaatattac 7260 agtag 7265 <210> 23 <211> 450 <212> PRT <213> Homo sapiens <400> 23 Met Gin Pro Pro Arg Glu Arg Leu Val Val Thr Gly Arg Ala Gly Trp 1 5 10 15 Met Gly Met Gly Arg Gly Ala Gly Arg Ser Ala Leu Gly Phe Trp Pro 20 25 30 Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro Ala Ala Thr Ser Pro Cys 35 40 45 Lys lie Leu Lys Cys Asn Ser Glu Phe Trp Ser Ala Thr Ser Gly Ser 50 55 60 His Ala Pro Ala Ser Asp Asp Thr Pro Glu Phe Cys Ala Ala Leu Arg 65 70 75 80 Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly Asp 85 90 95 Leu Ala Tyr His Ser Ala Val His Gly He Glu Asp Leu Met Ser Gin 100 105 110 His Asn Cys Ser Lys Asp Gly Pro Thr Ser Gin Pro Arg Leu Arg Thr 115 120 125 Leu Pro Pro Ala Gly Asp Ser Gin Glu Arg Ser Asp Ser Pro Glu lie 130 135 140 Cys His Tyr Glu Lys Ser Phe His Lys His Ser Ala Thr Pro Asn Tyr 145 150 155 160 15 2010 Thr His Cys Gly Leu Phe Gly Asp Pro His Leu Arg Thr Phe Thr Asp Jun 165 170 175 29 Arg Phe Gin Thr Cye Lys Val Gin Gly Ala Trp Pro Leu lie Asp Asn 180 185 190 Asn Tyr Leu Asn Val Gin Ala Thr Asn Thr Pro Val Leu Pro Gly Sex- 195 200 205 Ala Ala Thr Ala Thr Ser Lys Leu Thr lie He Phe Lys Asn Phe Gin 210 215 220 Glu Cys Val Asp Gin Lys Val Tyr Gin Ala Glu Met Asp Glu Leu Pro 225 230 235 240 2010202722 Ala Ala Phe Val Asp Gly Ser Lys Asn Gly Gly Asp Lys His Gly Ala 245 250 255 Asn Ser Leu Lys lie Thr Glu Lys Val Ser Gly Gin His Val Glu He 260 265 270 Gin Ala Lys Tyr He Gly Thr Thr lie Val Val Arg Gin Val Gly Arg 275 280 285 Tyr Leu Thr Phe Ala Val Arg Met Pro Glu Glu Val Val Asn Ala Val 290 295 300 Glu Asp Trp Asp Ser Gin Gly Leu Tyr Leu Cys Leu Arg Gly Cys Pro 305 310 315 320 Leu Asn Gin Gin He Asp Phe Gin Ala Phe His Thr Asn Ala Glu Gly 325 330 335 Thr Gly Ala Arg Arg Leu Ala Ala Ala Ser Pro Ala Pro Thr Ala Pro 340 345 350 Glu Thr Phe Pro Tyr Glu Thr Ala Val Ala Lys Cys Lys Glu Lys Leu 355 360 365 Pro Val Glu Asp Leu Tyr Tyr Gin Ala Cys Val Phe Asp Leu Leu Thr 370 375 380 Thr Gly Asp Val Asn Phe Thr Leu Ala Ala Tyr Tyr Ala Leu Glu Asp 385 390 395 400 Val Lys Met Leu Hie Ser Asn Lys Asp Lys Leu His Leu Tyr Glu Arg 405 410 415 Thr Arg Asp Leu Pro Gly Arg Ala Ala Ala Gly Leu Pro Leu Ala Pro 420 425 430 Arg Pro Leu Leu Gly Ala Leu Val Pro Leu Leu Ala Leu Leu Pro Val 435 440 445 Phe Cys 450 16 2010 Jun <210> 24 <211> 478 <212> PRT 29 <213> Homo sapiens’ <400> 24 Met He Arg Lys Lys Arg Lys Arg Ser Ala Pro Pro Gly Pro Cys Arg 722 1 5 10 15 (Μ Ο Ser His Gly Pro Arg Pro Ala Thr Ala Pro Ala Pro Pro Pro Ser Pro (Μ 20 25 30 Ο Glu Pro Thr Arg Pro Ala Trp Thr Gly Met Gly Leu Arg Ala Ala Pro Ο (Μ 35 40 45 Ser Ser Ala Ala Ala Ala Ala Ala Glu Val Glu Gin Arg Arg Ser Pro 50 55 60 Gly Leu Cys Pro Pro Pro Leu Glu Leu Leu Leu Leu Leu Leu Phe Ser 65 70 75 80 Leu Gly Leu Leu His Ala Gly Asp Cys Gin Gin Pro Ala Gin Cys Arg 85 90 95 lie Gin Lys Cys Thr Thr Asp Phe Val Ser Leu Thr Ser His Leu Asn 100 105 110 Ser Ala Val Asp Gly Phe Asp Ser Glu Phe Cys Lys Ala Leu Arg Ala 115 120 125 Tyr Ala Gly Cys Thr Gin Arg Thr Ser Lys Ala Cys Arg Gly Asn Leu 130 135 140 Val Tyr His Ser Ala Val Leu Gly He Ser Asp Leu Met Ser Gin Arg 145 150 155 160 Asn Cys Ser Lys Asp Gly Pro Thr Ser Ser Thr Asn Pro Glu Val Thr 165 170 175 His Asp Pro Cys Asn Tyr His Ser His Ala Gly Ala Arg Glu His Arg 180 185 190 Arg Gly Asp Gin Asn Pro Pro Ser Tyr Leu Phe Cys Gly Leu Phe Gly 195 200 205 Asp Pro His Leu Arg Thr Phe Lys Asp Asn Phe Gin Thr Cys Lys Val 210 215 220 Glu Gly Ala Trp Pro Leu lie Asp Asn Asn Tyr Leu Ser Val Gin Val 225 230 235 240 Thr Asn Val Pro Val Val Pro Gly Ser Ser Ala Thr Ala Thr Asn Lys 245 250 255 lie Thr lie He Phe Lys Ala His His Glu Cys Thr Asp Gin Lys Val 17 2010 260 265 270 Jun Tyr Gin Ala Val Thr Asp Asp Leu Pro Ala Ala Phe Val Asp Gly Thr 29 275 280 285 Thr Ser Gly Gly Asp Ser Asp Ala Lys Ser Leu Arg lie Val Glu Arg 290 295 300 Glu Ser Gly His Tyr Val Glu Met His Ala Arg Tyr lie Gly Thr Thr 305 310 315 320 Val Phe Val Arg Gin Val Gly Arg Tyr Leu Thr Leu Ala He Arg Met 325 330 335 Pro Glu Asp Leu Ala Met Ser Tyr Glu Glu Ser Gin Asp Leu Gin Leu 2010202722 340 345 350 Cys Val Asn Gly Cys Pro Leu Ser Glu Arg lie Asp Asp Gly Gin Gly 355 360 365 Gin Val Ser Ala lie Leu Gly His Ser Leu Pro Arg Thr Ser Leu Val 370 375 380 Gin Ala Trp Pro Gly Tyr Thr Leu Glu Thr Ala Asn Thr Gin Cys His 385 390 395 400 Glu Lys Met Pro Val Lys Asp lie Tyr Phe Gin Ser Cys Val Phe Asp 405 410 415 Leu Leu Thr Thr Gly Asp.Ala Asn Phe Thr Ala Ala Ala His Ser Ala 420 425 430 Leu Glu Asp Val Glu Ala Leu His Pro Arg Lys Glu Arg Trp His lie 435 440 445 Phe Pro Ser Ser Gly Asn Gly Thr Pro Arg Gly Gly Ser Asp Leu Ser 450 455 460 Val Ser Leu Gly Leu Thr Cys Leu He Leu. lie Val Phe Leu 465 470 475 <210> 25 <211> '420 <212> PRT <213> 1 Mus musculus <400> 25 Met Gly Gin Ser Pro Ser Pro Arg Ser Pro His Gly Ser Pro Pro Thr 1 5 10 15 Leu Ser Thr Leu Thr Leu Leu Leu Leu Leu .Cys Gly Gin Ala His Ser 20 25 30 Gin Cys Lys lie Leu Arg Cys Asn Ala Glu Tyr Val Ser Ser Thr Leu 18 2010 35 40 45 Jun Ser Leu Arg Gly Gly Gly Ser Pro Asp Thr Pro Arg Gly Gly Gly Arg 50 55 60 29 Gly Gly Leu Ala Ser Gly Gly Leu Cys Arg Ala Leu Arg Ser Tyr Ala 65 70 75 80 Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly Asp Leu Ala Phe 85 90 95 His Ser Ala Val His Gly He Glu Asp Leu Met lie Gin His Asn Cys 100 105 110 Ser Arg Gin Gly Pro Thr Ala Pro Pro Pro Ala Arg Gly Pro Ala Leu 2010202722 115 120 125 Pro Gly Ala Gly Pro Ala Pro Leu Thr Pro Asp Pro Cys Asp Tyr Glu 130 135 140 Ala Arg Phe Ser Arg Leu His Gly Arg Ala Pro Gly Phe Leu His Cys 145 150 155 160 Ala Ser Phe Gly Asp Pro His Val Arg Ser Phe His Asn Gin Phe His 165 170 175 Thr Cys Arg Val Gin Gly Ala Trp Pro Leu Leu Asp Asn Asp Phe Leu 180 185 190 Phe Val Gin Ala Thr Ser Ser Pro Val Ser Ser Gly Ala Asn Ala Thr 195 200 205 Thr lie Arg Lys He Thr He He Phe Lys Asn Met Gin Glu Cys He 210 215 220 Asp Gin Lys Val Tyr Gin Ala Glu Val Asp Asn Leu Pro Ala Ala Phe 225 230 235 240 Glu Asp Gly Ser He Asn Gly Gly Asp Arg Pro Gly Gly Ser Ser Leu 245 250 255 Ser He Gin Thr Ala Asn Leu Gly Ser His Val Glu lie Arg Ala Ala 260 265 270 Tyr lie Gly Thr Thr He He He Arg Gin Thr Ala Gly Gin Leu Ser 275 280 285 Phe Ser lie Arg Val Ala Glu Asp Val Ala Arg Ala Phe Ser Ala Glu 290 295 300 Gin Asp Leu Gin Leu Cys Val Gly Gly Cys Pro Pro Ser Gin Arg Leu- 305 310 315 320 Ser Arg Ser Glu Arg Asn Arg Arg Gly Ala He Ala lie Asp Thr Ala 325 330 335 19 2010 Arg Arg Leu Cys Lys Glu Gly Leu Pro Val Glu Asp Ala Tyr Phe Gin Jun 340 345 350 Ser Cys Val Phe Asp Val Ser Val Ser Gly Asp Pro Asn Phe Thr Val 29 355 360 365 Ala Ala Gin Thr Ala Leu Asp Asp Ala Arg He Phe Leu Thr Asp Leu 370 375 380 Glu Asn Leu His Leu Phe Pro Ser Asp Ala Gly Pro Pro Leu Ser Pro 385 390 395 400 Ala lie Cys Leu Val Pro Leu Leu Ser Ala Leu Phe Val Leu Trp Leu 405 410 415 2010202722 Cys Phe Ser Lys 420 <210> 26 <211> 422 <212> PRT <213> Rattus rattus <400> 26 Met Gly Asp Arg Gly Arg Ser Pro Ser Leu Arg Ser Pro His Gly Ser 1 5 10 15 Pro Pro Thr Leu Ser Thr Leu Thr Leu Leu Leu Leu Leu Cys Gly Gin 20 25 30 Ala His Ser Gin Cys Lys He Leu Arg Cys Asn Ala Glu Tyr Val Ser 35 40 45 Phe Thr Leu Ser Leu Arg Gly Gly Gly Ser Pro Asp Thr Pro Arg Gly 50 55 60 Gly Gly Arg Gly Gly Pro Ala Ser Gly Gly Leu Cys Arg Ala Leu Arg 65 70 75 80 Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly Asp 85 90 95 Leu Ala Phe His Ser Ala Val His Gly He Glu Asp Leu Met lie Gin 100 105 110 His Asn Cys Ser Arg Gin Gly Pro Thr Ala Ser Pro Pro Ala Arg Gly 115 120 12 5 Pro Ala Leu Pro Gly Ala Gly Pro Ala Pro Leu Thr Pro Asp Pro Cys 130 135 140 Asp Tyr Glu Ala Arg Phe Ser Arg Leu His Gly Arg Thr Pro Gly Phe 145 150 155 160 Leu His Cys Ala Ser Phe Gly Asp Pro His Val Arg Ser Phe His Asn 20 2010 165 170 175 Jun His Phe His Thr Cys Arg Val Gin Gly Ala Trp Pro Leu Leu Asp Asn 180 185 190 29 Asp Phe Leu Phe Val Gin Ala Thr Ser Ser Pro Val Ala Ser Gly Ala ι 195 200 205 Asn Ala Thr Thr lie Arg Lys He Thr He He Phe Lys Asn Met Gin 210 215 220 Glu Cys He Asp Gin Lys Val Tyr Gin Ala Glu Val Asp Asn Leu Pro 225 230 235 240 Ala Ala Phe Glu Asp Gly Ser Val Asn Gly Gly Asp Arg Pro Gly Gly 2010202722 245 250 255 Ser Ser Leu Ser lie Gin Thr Ala Asn Leu Gly Ser His Val Glu He 260 265 270 Arg Ala Ala Tyr He Gly Thr Thr He He val Arg Gin Thr Ala Gly 275 280 285 Gin Leu Ser Phe Ser lie Arg Val Ala Glu Asp Val Ala Arg Ala Phe 290 295 300 Ser Ala Glu Gin Asp Leu Gin Leu Cys Val Gly Gly Cys Pro Pro Ser 305 310 315 320 Gin Arg Leu Ser· Arg Ser Glu Arg Asn Arg Arg Gly Ala He Ala lie 325 330 335 Asp Thr Ala Arg Arg Leu Cys Lys Glu Gly Leu Pro Val Glu Asp Ala 340 345 350 Tyr Phe Gin Ser Cys Val Phe Asp Val Ser Val Ser Gly Asp Pro Asn 355 360 365 Phe Thr Val Ala Ala Gin Ser Ala Leu Asp Asp Ala Arg Val Phe Leu 370 375 380 Thr Asp Leu Glu Asn Leu His Leu Phe Pro Val Asp Ala Gly Pro Pro 385 390 395 400 Leu Ser Pro Ala Thr Cys Leu Val Arg Leu Leu Ser Val Leu Phe Val 405 410 415 Leu Trp Phe Cys lie Gin 420 <210> 27 <211> 366 <212> PRT <213> Fugu L 2010202722 29 Jun 2010 Glu Ser Pro His Arg Ser 225 Glu Leu Val Thr His Arg Ala Pro Pro Ser Pro Thr Leu 145 65 1 <400> Ala Ala Ala Gly Ala Arg Asp Arg 210 Lys Thr Gin Leu Gly Leu 130 Ser Glu Asp 50 Cys Ser 27 Ala Arg 275 Ser Phe Glu Arg Asp Arg Asp Ser Ala Ala 195 Phe Pro Ala lie 115 Thr Leu Cys 35 Val Gin Gly Gly Val Asn Asp 260 Ser Trp Gly Asp Val lie Val Arg 180 Gly Lys Ala Gly 100 20 Arg Leu Lys Pro Leu Gin Arg Asn Asn 245 Pro Thr Tyr Gin Arg 165 Cys Asn Pro 85 Ser 5 He Ala Glu Ala Leu Asp Ser His Met Asp 230 His Gin Leu Leu· Gly 150 Thr Met Ala Leu 70 Ser His Ala Gin Ala Ser Leu Thr Cys Ala His 215 Ala Phe Phe Tyr Ala Ala Arg 135 He 55 Glu Asp His Leu Val Cys 260 Thr Ser His Asp 200 Phe Thr Leu Cys Gin Arg Glu 120 Tyr Gly Cys 40 Asp Val Ala Pro Leu Ala Asp 265 Met Thr Gly Arg Asn He Tyr Asp Ala Pro Cys 185 105 Pro 25 Ala Gin Tyr Ala Asp Pro Val 250 Gin Gin Val Arg Arg Leu Cys Leu Arg Gly Gly Gly He Cys 90 Ser 170 10 21 Leu Leu Ser Thr Thr Gly Ala Arg Ala Asp Pro 235 Thr Gin Leu Phe Leu Pro 75 155 Leu Thr Val Cys Gin Ala Gly Ala Pro 220 Leu Lys Val Ser Leu Gly Leu 140 Phe He 60 Arg Pro Met Ala Val Asp Ala 285 Ser Gin Gin Asp Val Ser Tyr Thr His Ala 205 Pro 45 lie 125 Val Trp Ala Ser Val Gly Tyr Leu Ala 270 Ser Ser Gin Thr Pro Ser Pro Ser Pro 190 110 Pro 30 Arg Val Ala Thr Gly Gly Gly Ala His Glu Arg Ala Ala 255 Ser Val Leu Leu Tyr Ser 95 15 175 Asp Val Ala Arg Cys Gin Gly Glu Trp Thr Phe 240 Leu Cys Tyr Ser Ser Pro He 80 He 160 22 2010 Jun 290 295 300 Val Tyr His Gin Ala Cys lie Phe Asp Leu lie Thr Ser Gly Asp Leu 29 305 310 . 315 320 Asn Ser Ser Gly Ala Ala He Ser Ala Leu Gin Asp Ala Gin Lys Leu 325 330 335 He Ser Asp Pro Lys Arg Val His Leu Leu Ser Pro Thr Ser Ala Ala 340 345 350 Gin Arg Glu Asp His Leu Cys Leu Leu Leu Leu Leu Leu Ser 355 360 365 2010202722 <210> 28 <211> 432 <212> PRT <213 > Chicken <400> 28 Met Gly Arg Gly Ala Gly Ser Thr Ala Leu Gly Leu Phe Gin He Leu 1 5 10 15 Pro Val Phe Leu Cys He Phe Pro Pro Val Thr Ser Pro Cys Lys lie 20 25 30 Leu Lys Cys Asn Ser Glu Phe Trp Ala Ala Thr Ser Gly Ser His His 35 40 45 Leu Gly Ala Glu Glu Thr Pro Glu Phe Cys Thr Ala Leu Arg Ala Tyr 50 55 60 Ala His Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly Asp Leu Ala 65 70 75 80 Tyr His Ser Ala Val His Gly lie Asp Aep Leu Met Val Gin His Asn 85 90 95 Cys Ser Lys Asp Gly Pro Thr Ser Gin Pro Arg Leu Arg Thr Leu Pro 100 105 110 Pro Gly Asp Ser Gin Glu Arg Ser Asp Ser Pro Glu lie Cys His Tyr 115 120 125 Glu Lys Ser Phe His Lys His Ser Ala Ala Pro Asn Tyr Thr His Cys 130 135 140 Gly Leu Phe Gly Asp Pro His Leu Arg Thr Phe Thr Asp Thr Phe Gin 145 150 155 160 Thr Cys Lys Val Gin Gly Ala Trp Pro Leu lie Asp Asn Asn Tyr Leu 165 170 175 Asn Val Gin Val Thr Asn Thr Pro Val Leu Pro Gly Ser Ser Ala Thr 23 2010 180 185 190 Jun Ala Thr Ser Lys Leu Thr lie lie Phe Lys Ser Phe Gin Glu Cys Val 195 200 205 29 Glu Gin Lys Val Tyr Gin Ala Glu Met Asp Glu Leu Pro Ala Ala Phe 210 215 220 Ala Asp Gly Ser Lys Asn Gly Gly Asp Lys His Gly Ala Asn Ser Leu 225 230 235 240 Lys lie Thr Glu Lys Val Ser Gly Gin His lie Glu lie Gin Ala Lys 245 250 255 Tyr lie Gly Thr Thr lie Val Val Arg Gin Val Gly Arg Tyr Leu Thr 2010202722 260 265 270 Phe Ala Val Arg Met Pro Glu Glu Val Val Asn Ala Val Glu Asp Arg 275 280 285 Asp Ser Gin Gly Leu Tyr Leu Cys Leu Arg Gly Cys Pro Leu Asn Gin 290 295 300 Gin lie Asp Phe Gin Thr Phe Arg Leu Ala Gin Ala Ala Glu Gly Arg 305 310 315 320 Ala Arg Arg Lys Gly Pro Ser Leu Pro Ala Pro Pro Glu Ala Phe Thr 325 330 335 Tyr Glu Ser Ala Thr Ala Lys Cys Arg Glu Lys Leu Pro Val Glu Asp 340 345 350 Leu Tyr Phe Gin Ser Cys Val Phe Asp Leu Leu Thr Thr Gly Asp Val 355 360 365 Asn Phe Met Leu Ala Ala Tyr Tyr Ala Phe Glu Asp Val Lys Met Leu 370 375 380 His Ser Asn Lys Asp Lys Leu His Leu Tyr Glu Arg Thr Arg Ala Leu 385 390 395 400 Ala Pro Gly Asn Ala Ala Pro Ser Glu His Pro Trp Ala Leu Pro Ala 405 410 415 Leu Trp Val Ala Leu Leu Ser Leu Ser Gin Cys Trp Leu Gly Leu Leu 420 425 430 <210> 29 <211> 21 <212> DNA <213> Artificial <220> <223> Polynucleotide replication primer 24 2010 <400> 29 Jun tccaagtcag cgactctctc g 21 29 <210> 30 <211> 21 <212 > DNA <213> Artificial <220> <223> Polynucleotide replication primer <400> 30 tccaagtcag tgactctctc g 21 2010202722 <210> 31 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 31 acctgccgcg gggacctcgc c <210> 32 <211> 21 <212> DNA <213> Artificial <220> <223 > Fragment containing polymorphism <400> 32 acctgccgcg tggacctcgc c <210> 33 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 33 gcctgggaaa cctggctgga t <210> 34 <211> 21 <212> DNA <213> Artificial 25 2010 Jun <220> <223> Fragment containing polymorphism 29 <400> 34 gcctgggaaa gctggctgga t <210> 35 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism 2010202722 <400> 35 tcccttctgt ctttagctca t <210> 36 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 36 tcccttctgt gtttagctca t 21 <210> 37 <211> 20 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 37 gaggaggagg ccggggtgga <210> 38 <211> 23 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 38 gaggaggagg aggccggggt gga 23 26 2010 <210> 39 ' Jun <211> 21 <212> DNA <213> Artificial 29 <220> <223> Fragment containing polymorphism <400> 39 gcctccctgc cccggaccct t 21 <210> 40 <211> 21 <212> DNA 2010202722 <213> Artificial <220> <223> Fragment containing polymorphism <400> 40 gcctccctgc gccggaccct t 21 <210> 41 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 41 atggtcgtcc cccggggttc t 21 <210> 42 <211> 21 <212> DNA <213> Artificial . <220> <223> Fragment containing polymorphism <400> 42 atggtcgtcc accggggttc t 21 <210> 43 . <211> 21 <212> DNA . <213> Artificial <220> <223> Fragment containing polymorphism 27 2010 <400> 43 Jun cgtcccccgg ggttcttgca t 29 <210> 44 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 44 cgtcccccgg cgttcttgca t 2010202722 <210> 45 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 45 gtccaaggag cttggcctct a <210> 46 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 46 gtccaaggag attggcctct a <210> 47 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 47 cccccatggc gttgggggcc a <210> 48 <211> 21 <212> DNA <213> Artificial 28 2010 <220> Jun <223> Fragment containing polymorphism 29 <400> 48 cccccatggc tttgggggcc a 21 <210> 49 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism 2010202722 <400> 49 taagaacatg caggaatgca t 21 <210> 50 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 50 taagaacatg aaggaatgca t 21 <210> 51 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 51 gccttctcag ctgaacagga c 21 <210> 52 <211> 21 <212> DNA <213> Artificial <220> <223> Fragment containing polymorphism <400> 52 gccttctcag gtgaacagga c 21 <210> 53 <211> 21 29 2010 <212> DNA Jun <213> Artificial 29 <2,20> <223 > Fragment containing polymorphism <400> 53 agatgctggg gttcctcttt c 21 <210> 54 <211> 21 <212> DNA <213> Artificial 2010202722 <220> <223> Fragment containing polymorphism <400> 54 agatgctggg attcctcttt c 21 <210> 55 <211> 20 <212> DNA <213> Artificial <220> <223> Forward replication primer <400> 55 cacttgagcc caggaatttg <210> 56 <211> 20 <212> DNA <213> Artificial <220> <223> Reverse replication primer <400> 56 gactcactgc agccttgacc <210> 57 <211> 22 <212> DNA <213> Artificial <220> <223> Forward replication primer <400> 57 gtgtgctaca agtttgccga at 22 30 2010 Jun <210> 58 <211> 20 <212> DNA 29 <213> Artificial <220> <223> Reverse replication primer <400> 58 gcttgaaact gggagttgga <210> 59 <211> 22 2010202722 <212> DNA <213> Artificial <220> <223> Forward replication primer <400> 59 gggaaatggt cccataattc ct <210> 60 <211> 19 <212> DNA <213> Artificial <220> <223> Reverse replication primer <400> 60 cgccctgcca atatgttct <210> 61 <211> 22 <212> DNA <213> Artificial <220> <223> Forward replication primer <400> 61 ggtacttagc ctcgaaatga ga <21O> 62 <211> 20 <212> DNA <213> Artificial <220> <223> Reverse replication primer 2010202722 29 Jun 2010 gtgtcacaca <400> 62 actggttggt 31 20