U.S. patent application number 16/736208 was filed with the patent office on 2020-11-26 for diagnostic and prognostic test for sturge-weber syndrome, klippel-trenaunay-weber syndrome, and port-wine stains (pwss).
The applicant listed for this patent is DUKE UNIVERSITY, THE JOHNS HOPKINS UNIVERSITY, KENNEDY KRIEGER INSTITUTE. Invention is credited to Anne Comi, Douglas Marchuk, Jonathan Pevsner, Matthew Shirley.
Application Number | 20200370118 16/736208 |
Document ID | / |
Family ID | 1000005016192 |
Filed Date | 2020-11-26 |
United States Patent
Application |
20200370118 |
Kind Code |
A1 |
Pevsner; Jonathan ; et
al. |
November 26, 2020 |
DIAGNOSTIC AND PROGNOSTIC TEST FOR STURGE-WEBER SYNDROME,
KLIPPEL-TRENAUNAY-WEBER SYNDROME, AND PORT-WINE STAINS (PWSS)
Abstract
The present invention relates to the fields of neurological and
skin disorders. More specifically, the present invention provides
methods and compositions for diagnosing and prognosing Sturge-Weber
Syndrome (SWS), Klippel-Trenaunay-Weber Syndrome (KTWS), and Port
Wine Stains (PWS). In one embodiment, a method for prognosing or
monitoring treatment of a patient with SWS, KTWS and/or PWS
comprises the steps of (a) providing a sample from the patient
undergoing treatment; (b) determining the number of alleles in the
sample comprising at least one activating somatic mutation in the
guanine nucleotide-binding protein G subunit alpha (GNAQ) gene or
protein; (c) comparing the number of alleles comprising the at
least one somatic mutation to the number of alleles comprising the
somatic mutation from a patient sample provided prior to undergoing
treatment; and (d) determining that the patient is improving if
there is a decrease in the number of alleles comprising the at
least one somatic mutation from the sample of step (a).
Inventors: |
Pevsner; Jonathan;
(Baltimore, MD) ; Comi; Anne; (Baltimore, MD)
; Marchuk; Douglas; (Chapel Hill, NC) ; Shirley;
Matthew; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY
KENNEDY KRIEGER INSTITUTE
DUKE UNIVERSITY |
Baltimore
Baltimore
Durham |
MD
MD
NC |
US
US
US |
|
|
Family ID: |
1000005016192 |
Appl. No.: |
16/736208 |
Filed: |
January 7, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14784720 |
Oct 15, 2015 |
10544461 |
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PCT/US2014/034319 |
Apr 16, 2014 |
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16736208 |
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61812309 |
Apr 16, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/156 20130101; C12Q 2600/118 20130101 |
International
Class: |
C12Q 1/6883 20060101
C12Q001/6883 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with government support under grant
no. NS065705, awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-3. (canceled)
4. A method for treating a human patient having Sturge-Weber
Syndrome (SWS), Klippel-Trenaunay-Weber Syndrome (KTWS) or Port
Wine Stains (PWS) comprising the steps of: a. providing a nucleic
acid sample from the human patient; b. detecting whether the
somatic mutation c.548G>A in the guanine nucleotide-binding
protein G subunit alpha (GNAQ) gene is present in the nucleic acid
sample, wherein the detecting step is performed via whole genome
sequencing, whole exome sequencing or targeted amplicon sequencing;
c. identifying the human patient as having or likely to have SWS,
KTWS or PWS when the somatic mutation c.548G>A is present; and
d. administering an inhibitor of downstream effectors of the GNAQ
signaling pathway.
5. The method of claim 4, wherein the inhibitor is a protein kinase
C (PKC) inhibitor, a MEK inhibitor, a PI3k/AKT inhibitor, a
phospholipase C.beta. inhibitor or combinations thereof.
6-20. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 14/784,720, filed Oct. 15, 2015, now U.S. Pat.
No. 10,544,461, issued Jan. 28, 2020, which is a 35 U.S.C. .sctn.
371 U.S. national entry of International Application
PCT/US2014/034319 having an international filing date of Apr. 16,
2014, which claims the benefit of U.S. Provisional Application No.
61/812,309, filed Apr. 16, 2013, the content of each of the
aforementioned applications is herein incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the fields of neurologic,
ophthalmologic, orthopedic and skin disorders. More specifically,
the present invention provides methods and compositions for
diagnosing and prognosing Sturge-Weber Syndrome (SWS),
Klippel-Trenaunay-Weber Syndrome (KTWS), and Port Wine Stains
(PWS).
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0004] This application contains a sequence listing. It has been
submitted electronically via EFS-Web as an ASCII text file entitled
"P12119-04_ST25.txt." The sequence listing is 10,275 bytes in size,
and was created on Mar. 18, 2016. It is hereby incorporated by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0005] Sturge-Weber syndrome (SWS), also known as encephalofacial
angiomatosis, is a neurocutaneous disorder that occurs as a
sporadic, congenital condition characterized by a port-wine stain
(PWS) affecting the V1 territory of the face (the forehead and/or
eyelid) associated with a leptomengial angioma of the brain and
venous abnormalities of the eye. It occurs in both males and
females, in approximately 1 in 20-50,000 live births. Independently
occurring port-wine stains are much more common, occurring in
approximately 3 in 1000 births and commonly involve the head and
neck. A child born with a port-wine stain on the face has
approximately a 6% chance of having SWS, and this risk increases to
26% when the PWS is located in the V1 territory of the face.
Port-wine stains commonly have underlying soft and bony tissue
hypertrophy which may be mild or massive. When a port-wine
stain-associated hypertrophy involves a limb and has enlarged
venous or lymphatic vessels this is referred to as
Klippel-Trenaunay Weber syndrome (KTWS). KTWS has been reported in
association with SWS when the PWS is extensive and extends down on
to the trunk and affected limb. Therefore, it has been hypothesized
that SWS, KTWS, and PWS are likely to have the same underlying
somatic mutation(s). According to this hypothesis the precise
clinical manifestations are dependent upon where and when in the
developing fetus the somatic mutation occurs.
SUMMARY OF THE INVENTION
[0006] The present invention is based, at least in part, on the
discovery of an identifying mosaic somatic mutation in a specific
gene that can be used to detect, diagnose and prognose SWS, KTWS,
related neurocutaneous disorders and PWS.
[0007] Sturge-Weber syndrome (SWS) is a neurocutaneous disorder
that occurs as a sporadic, congenital condition. It is
characterized by a capillary malformation of the skin, i.e.,
port-wine stain (PWS) affecting the V1 distribution of the face
(forehead and/or eyelid) and abnormal capillary-venous vessels in
the leptomeninges of the brain (leptomeningeal angioma), sometimes
associated with abnormal venous vessels in the eye and glaucoma.
SWS is frequently associated with intractable seizures and
intellectual disability. Given its sporadic occurrence and the
scattered or asymmetric distribution of vascular malformations,
both PWS and SWS are hypothesized to be caused by a mosaic somatic
mutation that disrupts normal vascular development.
[0008] The present inventors tested this hypothesis by performing
whole genome sequencing of DNA from paired visibly affected and
normal tissue samples (n=3 SWS subjects). The presence of a somatic
mosaic mutation was assayed in 97 samples from 50 SWS, PWS, or
control subjects using amplicon sequencing and SNaPshot assays. The
effects of the mutation on downstream signaling were queried using
phosphorylation-specific antibodies for relevant effectors, and a
luciferase reporter assay.
[0009] The present inventors identified a non-synonymous
single-nucleotide variant (c.548G>A, p.Arg183Gln) in GNAQ in
samples of affected tissue from 88% (23/26) of subjects with SWS,
92% (12/13) of apparently non-syndromic PWS, none of the samples
affected tissue from four (0/4) subjects with an unrelated
cerebrovascular malformation, and no tissues from six (0/6) normal
subjects. Mutant allele prevalence in affected tissues ranged from
1% to 18.1%. Extracellular signal-regulated kinase (ERK) activity
was modestly increased during trans-expression of mutant GNAQ.
[0010] In one embodiment, diagnostic and prognostic detection of a
sequence mutation in Gnaq is performed by determining the number of
alleles in a biological sample having a sequence mutation in Gnaq.
In another embodiment, diagnostic and prognostic detection of a
sequence mutation in Gnaq is performed by determining the
percentage of wildtype alleles and mutant alleles (having a
sequence mutation in Gnaq) in DNA derived from a set of cells. In
another specific embodiment, a method for identifying a human
patient as having or likely to have Sturge-Weber syndrome (SWS)
comprises the steps of (a) providing a nucleic acid sample from the
human patient; (b) detecting whether the somatic mutation
c.548G>A in the guanine nucleotide-binding protein G subunit
alpha (GNAQ) gene is present in the nucleic acid sample; and (c)
identifying the human patient as having or likely have SWS when the
somatic mutation c.548G>A is present. In a more specific
embodiment, whether the mutation comprises an assessment of the
number of alleles in the sample comprising the mutation. For
example, the assessment can comprise determining the percentage of
mutatent alleles in the sample the comprise the mutation.
[0011] In certain embodiments, the detecting step is performed
using next generation sequencing. In particular embodiments, the
detecting step is performed using a single nucleotide primer
extension assay, whole genome sequencing, whole exome sequencing or
targeted amplicon sequencing. In certain embodiments, a detecting
step comprises performing polymerase chain reaction (PCR). In a
specific embodiment, the primers listed in SEQ ID NO:1 and SEQ ID
NO:2 are used. In other embodiments, the primers further comprise
SEQ ID NO:3 and SEQ ID NO:4.
[0012] In another aspect, the present invention provides methods
for treating SWS. In one embodiment, a method for treating a human
patient having SWS comprises the steps of (a) providing a nucleic
acid sample from the human patient; (b) detecting whether the
somatic mutation c.548G>A in the guanine nucleotide-binding
protein G subunit alpha (GNAQ) gene is present in the nucleic acid
sample, wherein the detecting step is performed via whole genome
sequencing, whole exome sequencing or targeted amplicon sequencing;
(c) identifying the human patient as having or likely have SWS when
the somatic mutation c.548G>A is present; and (d) administering
a GNAQ inhibitor to the human patient. In another embodiment, the
method further comprises administering to the human patient an
inhibitor of downstream effectors of the GNAQ signaling pathway. In
certain embodiments, the inhibitor is a protein kinase C (PKC)
inhibitor, a MEK inhibitor, a PI3k/AKT inhibitor, a phospholipase
C.beta. inhibitor or combinations thereof.
[0013] In a further aspect, the present invention provides methods
for monitoring treatment of a patient with Sturge-Weber syndrome
(SWS). In one embodiment, a method for monitoring treatment of a
patient with Sturge-Weber syndrome (SWS) comprises the steps of (a)
providing a sample from the patient undergoing treatment; (b)
determining the number of alleles in the sample comprising the
somatic mutation c.548G>A in the guanine nucleotide-binding
protein G subunit alpha (GNAQ) gene or p.R183Q in the GNAQ protein;
(c) comparing the number of alleles comprising the somatic mutation
to the number of alleles comprising the somatic mutation from a
patient sample provided prior to undergoing treatment; and (d)
determining that the patient is improving if there is a decrease in
the number of alleles comprising the somatic mutation from the
sample of step (a). In a further embodiment, the method further
comprises adjusting patient treatment based on step (d). In a
specific embodiment, the determining step comprises performing a
single nucleotide primer extension assay. In a more specific
embodiment, the assay is a snapshot assay. In another specific
embodiment, the primers used to perform the assay are SEQ ID NO:3
and SEQ ID NO:4.
[0014] In another embodiment, a method to monitor progress of SWS
treatment in a patient comprises (a) determining in a test sample
relative to normal sample, a somatic mutation in the GNAQ gene; (b)
repeating one or more times the determining step; and (c)
identifying an increase, decrease or stable level of the mutation
in the test sample over time. In a more specific embodiment, the
somatic mutation in the GNAQ gene is c.548G>A. In a further
embodiment a method for monitoring the response of patient with SWS
to therapy comprises the steps of (a) administering a therapy to
the patient; (b) obtaining a nucleic acid sample from the patient;
and (c) determining whether a somatic mutation at c.548G>A of
the GNAQ gene is present. In yet another embodiment, a method for
monitoring the response of patient with SWS to therapy comprises
the steps of (a) administering a therapy to the patient; (b)
obtaining a biological sample from the patient; and (c) determining
whether a somatic mutation at p.R183Q of the GNAQ protein is
present. In certain embodiments, the methods further comprise
adjusting treatment of the patient based on step (c).
[0015] In another aspect, the present invention provides methods
for treating or monitoring treatment of a patient with
Klippel-Trenaunay-Weber Syndrome (KTWS). In a specific embodiment,
a method for monitoring treatment of a patient with KTWS comprises
the steps of (a) providing a sample from the patient undergoing
treatment; (b) determining the number of alleles in the sample
comprising the somatic mutation c.548G>A in the guanine
nucleotide-binding protein G subunit alpha (GNAQ) gene or p.R183Q
in the GNAQ protein; (c) comparing the number of alleles comprising
the somatic mutation to the number of alleles comprising the
somatic mutation from a patient sample provided prior to undergoing
treatment; and (d) determining that the patient is improving if
there is a decrease in the number of alleles comprising the somatic
mutation from the sample of step (a). In a specific embodiment, the
determining step comprises performing a single nucleotide primer
extension assay. In a further embodiment, the method further
comprises adjusting patient treatment based on step (d).
[0016] In another embodiment, a method to monitor progress of KTWS
treatment in a patient comprises (a) determining in a test sample
relative to normal sample, a somatic mutation in the GNAQ gene; (b)
repeating one or more times the determining step; and (c)
identifying an increase, decrease or stable level of the mutation
in the test sample over time. In a more specific embodiment, the
somatic mutation in the GNAQ gene is c.548G>A. In a further
embodiment, a method for monitoring the response of patient with
KTWS to therapy comprises the steps of (a) administering a therapy
to the patient; (b) obtaining a nucleic acid sample from the
patient; and (c) determining whether a somatic mutation at
c.548G>A of the GNAQ gene is present. In a further embodiment, a
method for monitoring the response of patient with KTWS to therapy
comprises the steps of (a) administering a therapy to the patient;
(b) obtaining a biological sample from the patient; and (c)
determining whether a somatic mutation at p.R183Q of the GNAQ
protein is present.
[0017] In a further aspect, the present invention provides methods
for treating or monitoring treatment of progress of patient with
Port Wine Stains (PWS). In a specific embodiment, a method for
monitoring treatment of a patient with Port Wine Stains (PWS)
comprises the steps of (a) providing a sample from the patient
undergoing treatment; (b) determining the number of alleles in the
sample comprising the somatic mutation c.548G>A in the guanine
nucleotide-binding protein G subunit alpha (GNAQ) gene or p.R183Q
in the GNAQ protein; (c) comparing the number of alleles comprising
the somatic mutation to the number of alleles comprising the
somatic mutation from a patient sample provided prior to undergoing
treatment; and (d) determining that the patient is improving if
there is a decrease in the number of alleles comprising the somatic
mutation from the sample of step (a). In a further embodiment, the
method further comprises adjusting patient treatment based on step
(d).
[0018] In another embodiment, a method for prognosing or monitoring
treatment of a patient with SWS, KTWS and/or PWS comprises the
steps of (a) providing a sample from the patient undergoing
treatment; (b) determining the number of alleles in the sample
comprising at least one activating somatic mutation in the guanine
nucleotide-binding protein G subunit alpha (GNAQ) gene or protein;
(c) comparing the number of alleles comprising the at least one
somatic mutation to the number of alleles comprising the somatic
mutation from a patient sample provided prior to undergoing
treatment; and (d) determining that the patient is improving if
there is a decrease in the number of alleles comprising the at
least one somatic mutation from the sample of step (a). In a
further embodiment, the method further comprises adjusting patient
treatment based on step (d). In another embodiment, the at least
one activating somatic mutation in the GNAQ gene or protein is
c.548G>A or p.R183Q, respectively.
[0019] In another aspect, the present invention provides methods
and compositions useful for treating PWS and/or SWS. In certain
embodiments, the methods and compositions utilize a GNAQ modulator.
In one embodiment, the modulator is an antagonist or inhibitor of
GNAQ. In an alternative embodiment, the modulator is an agonist of
GNAQ. In specific embodiments, the GNAQ antagonist is a small
molecule, an antibody, or an inhibitory nucleic acid molecule
(e.g., siRNA, antisense, or shRNA).
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1A-1F: Photographs and MRI images from representative
individuals with Sturge-Weber syndrome or isolated port-wine stain.
(FIG. 1A, 1B) Facial PWS birthmark photographed at birth in subject
37; note the left-sided V1 distribution. Child began having
seizures at 7 months of life. (FIG. 1C) Isolated PWS birthmark of
the left shoulder from subject 11 (see Table 1); birthmark is flat
and red without evidence of hypertrophy or cobble-stoning or any
other associated vascular or lymphatic anomaly. (FIG. 1D-1F) Axial
contrast-enhanced MRI imaging of the brain of a representative
individual demonstrating left-sided hemispheric leptomeningeal
enhancement (white arrows), an enlarged and enhancing left-sided
choroid plexus (red arrow) and left hemispheric brain atrophy
(yellow arrows).
[0021] FIG. 2A-2F: Downstream effectors of GNAQ. Wild-type GNAQ,
p.Arg183Gln and p.Gln209Leu were transfected into HEK 293T cells.
(FIG. 2A) Strongly increased phosphorylation of ERK is seen with
the GNAQ p.Gln209Leu and weaker but statistically significant
activation with GNAQ p.Arg183Gln. (FIG. 2B) Increased
phosphorylation of p38 with GNAQ p.Gln209Leu is seen but not with
GNAQ p.Arg183Gln. (FIG. 2C) Increased phosphorylation of JNK is
seen with GNAQ p.Gln209Leu and weaker activation with GNAQ
p.Arg183Gln. (FIG. 2D) No change in phosphorylation of AKT is seen
with either the GNAQ p.Arg183Gln or p.Gln209Leu constructs. (FIG.
2E) Control for transfection efficiency showing comparable amounts
of the three transfected, FLAG-tagged proteins were transfected
into HEK 293T cells. (FIG. 2F) SRE luciferase assay. Relative
luciferase activity expressed under the control of the SRE
promoter, coexpressed with GNAQ p.Arg183Gln, p.Gln209Leu, or
wild-type constructs. (* p<0.05) (.dagger.p=0.052)
Abbreviations: A.U--arbitrary units.
[0022] FIG. 3: Simulation of variant detection and resulting
detection probabilities at varying read depth. Simulation was
performed using 1,000,000 draws from an allele pool with 1% mutant
allele frequency at read depths from 1 to 10,192 using a detection
threshold for genotype determination of 0.5% mutant allele.
DETAILED DESCRIPTION OF THE INVENTION
[0023] It is understood that the present invention is not limited
to the particular methods and components, etc., described herein,
as these may vary. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention. It must be noted that as used herein and in the
appended claims, the singular forms "a," "an," and "the" include
the plural reference unless the context clearly dictates otherwise.
Thus, for example, a reference to a "protein" is a reference to one
or more proteins, and includes equivalents thereof known to those
skilled in the art and so forth.
[0024] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Specific
methods, devices, and materials are described, although any methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention.
[0025] All publications cited herein are hereby incorporated by
reference including all journal articles, books, manuals, published
patent applications, and issued patents. In addition, the meaning
of certain terms and phrases employed in the specification,
examples, and appended claims are provided. The definitions are not
meant to be limiting in nature and serve to provide a clearer
understanding of certain aspects of the present invention.
I. Definitions
[0026] The term "Gnaq" refers to the alpha subunit of a guanine
nucleotide binding protein (G-protein). The term encompasses
nucleic acid and polypeptide polymorphic variants, alleles,
mutants, and fragments of Gnaq. Such sequences are well known in
the art. Exemplary human Gnaq sequences are available under the
reference sequences NM_002072 in the NCBI nucleotide database
(nucleotide sequence) (and NM_002072.3) and accession number NP
002063.2 (polypeptide sequence). The sequence NM_002072 is provided
as SEQ ID NO:12 as an exemplary nucleotide sequence. The exemplary
polypeptide sequence is shown in SEQ ID NO:13.
[0027] A "Gnaq-dependent mutation" as used in the context of this
application refers to cells that have a defect in Gnaq that
activates or otherwise disrupts the function of Gnaq, i.e., has an
"activating" mutation, in comparison to cells that do not have the
mutation, and leads to a loss or decrease of GTP hydrolyzing
activity of the mutant G-.alpha. subunit. The Gnaq mutation, e.g.,
a substitution mutation, can result in constitutive activity of the
protein. The "Gnaq-dependent mutation" may have one or more of such
mutations, e.g., the cells may have somatic substitution mutation
involving R183. A "Gnaq-dependent mutation" may also have mutations
in genes other than Gnaq.
[0028] The terms "sample," "patient sample," "biological sample,"
and the like, encompass a variety of sample types obtained from a
patient, individual, or subject and can be used in a diagnostic,
prognostic or monitoring assay. The patient sample may be obtained
from a healthy subject, a diseased patient including, for example,
a patient having associated symptoms of SWS, KTWS or PWS. Moreover,
a sample obtained from a patient can be divided and only a portion
may be used for diagnosis, prognosis or monitoring. Further, the
sample, or a portion thereof, can be stored under conditions to
maintain sample for later analysis. The definition specifically
encompasses blood and other liquid samples of biological origin
(including, but not limited to, peripheral blood, serum, plasma,
urine, saliva, amniotic fluid, stool and synovial fluid), solid
tissue samples such as a biopsy specimen or tissue cultures or
cells derived therefrom and the progeny thereof. In a specific
embodiment, a sample comprises a skin sample. In another
embodiment, a sample of brain tissue is used. In other embodiments,
a sample comprises a blood or serum sample. The definition also
includes samples that have been manipulated in any way after their
procurement, such as by centrifugation, filtration, precipitation,
dialysis, chromatography, treatment with reagents, washed, or
enriched for certain cell populations. The terms further encompass
a clinical sample, and also include cells in culture, cell
supernatants, tissue samples, organs, and the like. Samples may
also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded
tissue blocks, such as blocks prepared from clinical or
pathological biopsies, prepared for pathological analysis or study
by immunohistochemistry.
[0029] The terms "providing a sample" and "providing a biological
(or patient) sample" are used interchangeably and mean to provide
or obtain a biological sample for use in methods described in this
invention. Most often, this will be done by removing a sample of
cells from a patient, but can also be accomplished by using
previously isolated cells (e.g., isolated by another person, at
another time, and/or for another purpose), or by performing the
methods of the invention in vivo. Archival tissues, having
treatment or outcome history, can also be used.
[0030] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site
www.ncbi.nlm nih gov/BLAST/ or the like). Such sequences are then
said to be "substantially identical." This definition also refers
to, or may be applied to, the complement of a test sequence. The
definition also includes sequences that have deletions and/or
additions, as well as those that have substitutions, as well as
naturally occurring, e.g., polymorphic or allelic variants, and
man-made variants. As described below, the preferred algorithms can
account for gaps and the like. Preferably, identity exists over a
region that is at least about 25 amino acids or nucleotides in
length, or more preferably over a region that is 50-100 amino acids
or nucleotides in length. For example, a nucleic acid probe that is
used in the invention, may have at least 85%, typically 90%, or
95%, sequence identity to a contiguous region of SEQ ID NO:12.
[0031] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0032] A "comparison window", as used herein, includes reference to
a segment of one of the number of contiguous positions selected
from the group consisting typically of from 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequences for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local alignment algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson &
Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0033] Examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity include the BLAST
and BLAST 2.0 algorithms, which are described in Altschul et al.,
Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol.
Biol. 215:403-410 (1990). For the purposes of this invention, BLAST
and BLAST 2.0 are used with default parameters to determine percent
sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(NCBI). The BLASTN program (for nucleotide sequences) uses as
defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff
of 100, match score=2, mismatch score=-3, and a comparison of both
strands. For amino acid (protein) sequences, the BLASTP program
uses as defaults a wordlength (W) of 3, an expectation (E) of 10,
and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915)). For the purposes of this
invention, the BLAST2.0 algorithm is used with the default
parameters and the filter off.
[0034] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, e.g.,
where the two peptides differ only by conservative substitutions.
Another indication that two nucleic acid sequences are
substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequences.
[0035] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components that normally accompany it as found in its native state.
Purity and homogeneity are typically determined using analytical
chemistry techniques such as polyacrylamide gel electrophoresis or
high performance liquid chromatography. A protein or nucleic acid
that is the predominant species present in a preparation is
substantially purified. In particular, an isolated nucleic acid is
separated from some open reading frames that naturally flank the
gene and encode proteins other than protein encoded by the gene.
The term "purified" in some embodiments denotes that a nucleic acid
or protein gives rise to essentially one band in an electrophoretic
gel. In certain embodiments, it means that the nucleic acid or
protein is at least 85% pure, at least 95% pure, and at least 99%
pure. "Purify" or "purification" in other embodiments means
removing at least one contaminant from the composition to be
purified. In this sense, purification does not require that the
purified compound be homogenous, e.g., 100% pure.
[0036] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers, those containing modified
residues, and non-naturally occurring amino acid polymer.
[0037] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, e.g., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs may have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0038] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0039] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical or associated, e.g.,
naturally contiguous, sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode most proteins. For instance, the codons GCA, GCC, GCG
and GCU all encode the amino acid alanine. Thus, at every position
where an alanine is specified by a codon, the codon can be altered
to another of the corresponding codons described without altering
the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, often silent variations of a nucleic acid
which encodes a polypeptide is implicit in a described sequence
with respect to the expression product, but not with respect to
actual probe sequences.
[0040] As to amino acid sequences, one of ordinary skill in the art
recognizes that individual substitutions, deletions or additions to
a nucleic acid, peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" where the alteration results in the substitution
of an amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Such conservatively modified variants are in
addition to and do not exclude polymorphic variants, interspecies
homologs, and alleles of the invention. Typical conservative
substitutions for one another: 1) Alanine (A), Glycine (G); 2)
Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine
(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),
Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),
Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0041] "Nucleic acid" or "oligonucleotide" or "polynucleotide" or
grammatical equivalents used herein means at least two nucleotides
covalently linked together. Oligonucleotides are typically from
about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides
in length, up to about 100 nucleotides in length. Nucleic acids and
polynucleotides are polymers of any length, including longer
lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000,
etc. A nucleic acid of the present invention will generally contain
phosphodiester bonds, although in some cases, nucleic acid analogs
are included that may have alternate backbones, comprising, e.g.,
phosphoramidate, phosphorothioate, phosphorodithioate, or
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides
and Analogues: A Practical Approach, Oxford University Press); and
peptide nucleic acid backbones and linkages. Other analog nucleic
acids include those with positive backbones; non-ionic backbones,
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, Carbohydrate Modifications in Antisense Research,
Sanghui & Cook, eds. Nucleic acids containing one or more
carbocyclic sugars are also included within one definition of
nucleic acids. Modifications of the ribose-phosphate backbone may
be done for a variety of reasons, e.g., to increase the stability
and half-life of such molecules in physiological environments or as
probes on a biochip. Mixtures of naturally occurring nucleic acids
and analogs can be made; alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made.
[0042] The nucleic acids may be single stranded or double stranded,
as specified, or contain portions of both double stranded or single
stranded sequence. As will be appreciated by those in the art, the
depiction of a single strand also defines the sequence of the
complementary strand; thus the sequences described herein also
provide the complement of the sequence. Unless otherwise indicated,
a particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (e.g., degenerate codon
substitutions) and complementary sequences, as well as the sequence
explicitly indicated. The nucleic acid may be DNA, both genomic and
cDNA, RNA or a hybrid, where the nucleic acid may contain
combinations of deoxyribo- and ribo-nucleotides, and combinations
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
"Transcript" typically refers to a naturally occurring RNA, e.g., a
pre-mRNA, hnRNA, or mRNA. As used herein, the term "nucleoside"
includes nucleotides and nucleoside and nucleotide analogs, and
modified nucleosides such as amino modified nucleosides. In
addition, "nucleoside" includes non-naturally occurring analog
structures. Thus, e.g. the individual units of a peptide nucleic
acid, each containing a base, are referred to herein as a
nucleoside.
[0043] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include .sup.32P, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins or other entities which can be
made detectable, e.g., by incorporating a radiolabel into the
peptide or used to detect antibodies specifically reactive with the
peptide. The labels may be incorporated into the KIT nucleic acids,
proteins and antibodies at any position. Any method known in the
art for conjugating the antibody to the label may be employed,
e.g., using methods described in Hermanson, Bioconjugate Techniques
1996, Academic Press, Inc., San Diego.
[0044] A "labeled nucleic acid probe or oligonucleotide" is one
that is bound, either covalently, through a linker or a chemical
bond, or noncovalently, through ionic, van der Waals,
electrostatic, or hydrogen bonds to a label such that the presence
of the probe may be detected by detecting the presence of the label
bound to the probe. Alternatively, method using high affinity
interactions may achieve the same results where one of a pair of
binding partners binds to the other, e.g., biotin,
streptavidin.
[0045] As used herein a "nucleic acid probe or oligonucleotide" is
defined as a nucleic acid capable of binding to a target nucleic
acid of complementary sequence through one or more types of
chemical bonds, usually through complementary base pairing, usually
through hydrogen bond formation. As used herein, a probe may
include natural (i.e., A, G, C, or T) or modified bases
(7-deazaguanosine, inosine, etc.). In addition, the bases in a
probe may be joined by a linkage other than a phosphodiester bond,
so long as it does not functionally interfere with hybridization.
Thus, e.g., probes may be peptide nucleic acids in which the
constituent bases are joined by peptide bonds rather than
phosphodiester linkages. It will be understood by one of skill in
the art that probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the
stringency of the hybridization conditions. The probes are
preferably directly labeled as with isotopes, chromophores,
lumiphores, chromogens, or indirectly labeled such as with biotin
to which a streptavidin complex may later bind. By assaying for the
presence or absence of the probe, one can detect the presence or
absence of the select sequence or subsequence. Diagnosis or
prognosis may be based at the genomic level, or at the level of RNA
or protein expression.
[0046] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, e.g., recombinant cells
express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all. By the term "recombinant nucleic acid" herein is meant nucleic
acid, originally formed in vitro, in general, by the manipulation
of nucleic acid, e.g., using polymerases and endonucleases, in a
form not normally found in nature. Similarly, a "recombinant
protein" is a protein made using recombinant techniques, i.e.,
through the expression of a recombinant nucleic acid as depicted
above.
[0047] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule to a
particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a mixture (e.g., total
cellular or library DNA or RNA, an amplification reaction), such
that the binding of the molecule to the particular nucleotide
sequence is determinative of the presence of the nucleotide
sequence is the mixture.
[0048] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, preferably 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. For PCR, a temperature of about 36.degree. C.
is typical for low stringency amplification, although annealing
temperatures may vary between about 32.degree. C. and 48.degree. C.
depending on primer length. For high stringency PCR amplification,
a temperature of about 62.degree. C. is typical, although high
stringency annealing temperatures can range from about 50.degree.
C. to about 65.degree. C., depending on the primer length and
specificity. Typical cycle conditions for both high and low
stringency amplifications include a denaturation phase of
90.degree. C.-95.degree. C. for 30 sec-2 min., an annealing phase
lasting 30 sec. to 2 min., and an extension phase of about
72.degree. C. for 1-2 min. Protocols and guidelines for low and
high stringency amplification reactions are provided, e.g., in
Innis et al. (1990) PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc. N.Y.).
[0049] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al.
[0050] The phrase "functional effects" in the context of assays for
testing compounds that inhibit activity of a Gnaq protein includes
the determination of a parameter that is indirectly or directly
under the influence of the Gnaq protein or nucleic acid, e.g., a
functional, physical, or chemical effect, such as the ability to
alter GTP hydrolase activity. Activities or functional effect of
Gnaq can include protein-protein interaction activity, e.g., the
ability of Gnaq to bind an antibody or other protein with which it
interacts; GTP hydrolase activity, the ability of Gnaq to bind GTP
and/or GDP; contact inhibition and density limitation of growth;
cellular proliferation; cellular transformation; changes in
pigmentation; growth factor or serum dependence; and mRNA and
protein expression in cells. "Functional effects" include in vitro,
in vivo, and ex vivo activities.
[0051] As used herein, "inhibitors" or "antagonists" of Gnaq (e.g.
"Gnaq antagonists") refer to modulatory molecules or compounds
that, e.g., bind to, partially or totally block activity, decrease,
prevent, delay activation, inactivate, desensitize, or down
regulate the activity or expression of Gnaq protein, phospholipase
CP, or downstream molecules regulated by Gnaq, e.g., protein kinase
C (PKC). In a specific embodiment, an inhibitor specifically
inhibits the mutant Gnaq described herein. In such embodiments,
exogenous wild-type Gnaq can be administered in conjunction with
the specific Gnaq inhibitor. Inhibitors can include siRNA or
antisense RNA, genetically modified versions of Gnaq protein, e.g.,
versions with altered activity, as well as naturally occurring and
synthetic Gnaq antagonists, antibodies, small chemical molecules
and the like. Gnaq inhibitors for use in the invention are known in
the art. For example, non-limiting exemplary inhibitors suitable
for use with the present invention can include inhibitors of PKC,
for example the relatively nonspecific PKC inhibitor staurosporine,
the staurosporie analogue CPG41251, bryostatin-1, KAI-9803,
7-hydroxystaurosporine, L-threo-dihydrosphingosine (safingol), the
non-selective PKC inhibitor (PKC412), ilmofosine (BM 41 440),
indolcarbazole Go6796 which is a more specific inhibitor of the
classical PKC isoforms including PKC.mu., the PKC-alpha antisense
inhibitor LY900003, and the PKC-beta inhibitors LY333531, LY317615
(Enzastaurin). Non-limiting exemplary inhibitors of phospholipase
C.beta. can include edelfosine and fluvirusin B[2]. Assays for
identifying other inhibitors can be performed in vitro or in vivo,
e.g., in cells, or cell membranes, by applying test inhibitor
compounds, and then determining the functional effects on
activity.
[0052] In some embodiments, samples or assays comprising Gnaq
proteins that are treated with a potential inhibitor are compared
to control samples without the inhibitor, to examine the effect on
activity. Typically, control samples, e.g., cells, that have a Gnaq
mutation and that are untreated with inhibitors are assigned a
relative protein activity value of 100%. Inhibition of Gnaq is
achieved when the activity value relative to the control is changed
at least about 20%, at least about 50%, at least about 75-100%, or
more. In some embodiments, an inhibitor will activate a particular
activity, such as GTP hydrolysis, however, the net effect will be a
decrease in the activity of Gnaq, e.g., in comparison to controls
that have activated Gnaq.
[0053] As used herein, "antibody" includes reference to an
immunoglobulin molecule immunologically reactive with a particular
antigen, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies) and
heteroconjugate antibodies (e.g., bispecific antibodies). The term
"antibody" also includes antigen binding forms of antibodies,
including fragments with antigen-binding capability (e.g., Fab',
F(ab').sub.2, Fab, Fv and rIgG. See also, Pierce Catalog and
Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.). See
also, e.g., Kuby, J., Immunology, 3.sup.rd Ed., W.H. Freeman &
Co., New York (1998). The term also refers to recombinant single
chain Fv fragments (scFv). The term antibody also includes bivalent
or bispecific molecules, diabodies, triabodies, and tetrabodies.
Bivalent and bispecific molecules are described in, e.g., Kostelny
et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992)
Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.
(1994) J Immunol: 5368, Zhu et al. (1997) Protein Sci 6:781, Hu et
al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res.
53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.
[0054] An antibody immunologically reactive with a particular
antigen can be generated by recombinant methods such as selection
of libraries of recombinant antibodies in phage or similar vectors,
see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al.,
Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech.
14:309-314 (1996), or by immunizing an animal with the antigen or
with DNA encoding the antigen.
[0055] Typically, an immunoglobulin has a heavy and light chain
Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). Light
and heavy chain variable regions contain four framework" regions
interrupted by three hypervariable regions, also called
complementarity-determining regions (CDRs).
[0056] References to "V.sub.H" or a "VH" refer to the variable
region of an immunoglobulin heavy chain of an antibody, including
the heavy chain of an Fv, scFv, or Fab. References to "V.sub.L" or
a "VL" refer to the variable region of an immunoglobulin light
chain, including the light chain of an Fv, scFv, dsFv or Fab.
[0057] A "chimeric antibody" is an immunoglobulin molecule in which
(a) the constant region, or a portion thereof, is altered, replaced
or exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0058] A "humanized antibody" is an immunoglobulin molecule which
contains minimal sequence derived from non-human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient
antibody) in which residues from a complementary determining region
(CDR) of the recipient are replaced by residues from a CDR of a
non-human species (donor antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some
instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human residues. Humanized antibodies
may also comprise residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. In
general, a humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the
framework (FR) regions are those of a human immunoglobulin
consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin (Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
Humanization can be essentially performed following the method of
Winter and co-workers (Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species.
[0059] The term "fully human antibody" refers to an immunoglobulin
comprising human hypervariable regions in addition to human
framework and constant regions. Such antibodies can be produced
using various techniques known in the art. For example in vitro
methods involve use of recombinant libraries of human antibody
fragments displayed on bacteriophage (e.g., McCafferty et al.,
1990, Nature 348:552-554; Hoogenboom & Winter, J. Mol. Biol.
227:381 (1991); and Marks et al., J. Mol. Biol. 222:581 (1991)),
yeast cells (Boder and Wittrup, 1997, Nat Biotechnol 15:553-557),
or ribosomes (Hanes and Pluckthun, 1997, Proc Natl Acad Sci USA
94:4937-4942). Similarly, human antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, e.g., in U.S. Pat.
Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications:
(e.g., Jakobavits, Adv Drug Dehv Rev. 31:33-42 (1998), Marks et
al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0060] "Epitope" or "antigenic determinant" refers to a site on an
antigen to which an antibody binds. Epitopes can be formed both
from contiguous amino acids or noncontiguous amino acids juxtaposed
by tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance See,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed (1996).
II. GNAQ Mutations
[0061] In one aspect of the invention, the presence of an
activating mutation in a Gnaq polynucleotide, e.g., mRNA or genomic
DNA, or increased activity of a Gnaq protein and/or the presence of
a sequence mutation in the Gnaq protein, is determined in
biological samples. In some embodiments activating mutations in
Gnaq nucleic acids are determined. As noted, human Gnaq sequences
are well known. The Gnaq gene maps to 9q21 and the mRNA transcript
is 2.188 kb, which encodes a 359 amino acid protein.
[0062] "Sequence mutation" as used herein refers to changes in a
polynucleotide sequence that result in changes to protein activity.
Mutations can be nucleotide substitutions, such as single
nucleotide substitutions, insertions, or deletions. Gnaq mutations
are typically activating mutations that lead to constitutive
activation of Gnaq activity. Without being bound to a theory, it is
believed that the constitutive activity results from a lack of the
GTP-hydrolase activity in the mutant Gnaq protein.
[0063] The present invention is based in part on the discovery of
heterozygous somatic activating mutations present in Gnaq in
patients with SWS, KTWS and PWS. A mutation may be in any part of
the Gnaq gene where the mutation leads to activation of Gnaq. In
one embodiment, the mutation 547C>T results in an amino acid
substitution, R183Q.
[0064] In the present invention, an altered level of Gnaq activity
and/or a sequence mutation in Gnaq is detected for the diagnosis,
prognosis or treatment monitoring of patients with SWS, KTWS, or
PWS. Thus, biological samples obtained from patients that have SWS,
KTWS and/or PWS can be analyzed for mutations in the sequence of
Gnaq DNA, mRNA or protein. The presence of a mutation is
conveniently analyzed using samples of RNA, DNA, or protein.
[0065] 1. Detection of GNAQ Sequence Mutations
[0066] In one embodiment, diagnostic and prognostic detection of a
sequence mutation in Gnaq is performed by determining the number of
alleles in a biological sample having a sequence mutation in Gnaq.
In another embodiment, diagnostic and prognostic detection of a
sequence mutation in Gnaq is performed by determining the
percentage of wildtype alleles and mutant alleles (having a
sequence mutation in Gnaq) in DNA derived from a set of cells.
Methods of evaluating the sequence of a particular gene are well
known to those of skill in the art, and include, inter alia,
hybridization and amplification based assays. In a specific
embodiment, sequence mutation in Gnaq can be determined using a
probe that selectively hybridizes to the mutant sequence. In other
embodiments, sequencing can be used to identify the Gnaq mutation
in cells.
[0067] In some embodiments, a Gnaq sequence mutation in a
biological sample is determined by in situ hybridization, e.g.,
fluorescence in situ hybridization. In situ hybridization assays
are well known (e.g., Angerer (1987) Meth. Enzymol 152: 649). The
probes used in such applications specifically hybridize to the
region of the Gnaq sequence harboring the mutation. Probes are
sufficiently long, e.g., from about 10, 15, or 20 nucleotides to
about 50 or more nucleotides, so as to specifically hybridize with
the target nucleic acid(s) under stringent conditions.
[0068] Any of a number of other hybridization-based assays can be
used to detect a sequence mutation in Gnaq in the cells of a
biological sample. For example, dot blots, array-based assays and
the like can be used to determine Gnaq sequence mutations.
[0069] In some embodiments, amplification-based assays are used to
detect sequence mutations in Gnaq or to measure the levels of Gnaq
transcript. In such an assay, the Gnaq nucleic acid sequence acts
as a template in an amplification reaction (e.g., Polymerase Chain
Reaction, or PCR). Exemplary amplification-based assays can include
RT-PCR methods well known to the skilled artisan (see, e.g.,
Ausubel et al., supra). Detailed protocols for PCR of DNA and RNA,
including quantitative amplification methods, are known (see, e.g.,
Innis et al. (1990) PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc. N.Y.; and Ausubel and Russell
& Sambrook, both supra). The known nucleic acid sequences for
Gnaq (see, e.g., SEQ ID NO:12) are sufficient to enable one of
skill to routinely select primers to amplify any portion of the
gene. Suitable primers for amplification of specific sequences can
be designed using principles well known in the art (see, e.g.,
Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual
(1995)).
[0070] Other suitable amplification methods include, but are not
limited to, ligase chain reaction (LCR) (see, Wu and Wallace (1989)
Genomics 4: 560, Landegren et al. (1988) Science 241:1077, and
Barringer et al. (1990) Gene 89: 117), transcription amplification
(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173),
self-sustained sequence replication (Guatelli et al. (1990) Proc.
Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR,
etc.
[0071] The presence of mutations in Gnaq DNA or RNA sequences can
be determined using any technique known in the art. For example, in
one embodiment, allele-specific oligonucleotide hybridization may
be used, which relies on distinguishing a mutant from a normal
nucleic acid sequence using an oligonucleotide that specifically
hybridizes to the mutant or normal nucleic acid sequence. This
method typically employs short oligonucleotides, e.g., 15-20
nucleotides, in length, that are designed to differentially
hybridize to the normal or mutant allele. Guidance for designing
such probes is available in the art. The presence of a mutant
allele is determined by measuring the amount of allele-specific
oligonucleotide that hybridizes to the sample
[0072] Suitable assay formats for detecting hybrids formed between
probes and target nucleic acid sequences in a sample are known in
the art and include the immobilized target (dot-blot) format and
immobilized probe (reverse dot-blot or line-blot) assay formats.
Dot blot and reverse dot blot assay formats are described in U.S.
Pat. Nos. 5,310,893; 5,451,512; 5,468,613; and 5,604,099.
[0073] In other embodiments, the presence (or amount) of a normal
or mutant Gnaq nucleic acid can be detected using allele-specific
amplification or primer extension methods. These reactions
typically involve use of primers that are designed to specifically
target a normal or mutant allele via a mismatch at the 3' end of a
primer. The presence of a mismatch affects the ability of a
polymerase to extend a primer when the polymerase lacks
error-correcting activity. The amount of amplified product can be
determined using a probe or by directly measuring the amount of DNA
present in the reaction.
[0074] Detection of levels of Gnaq nucleic acids, e.g., levels of
normal and/or mutant Gnaq polynucleotides, or the presence of a
Gnaq mutation can also be performed using a quantitative assay such
as a 5'-nuclease activity (also referred to as a "TaqMan.RTM."
assay), e.g., as described in U.S. Pat. Nos. 5,210,015; 5,487,972;
and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad. Sci. USA
88:7276-7280. In such an assay, labeled detection probes that
hybridize within the amplified region are added during the
amplification reaction. In some embodiments, the hybridization
probe can be an allele-specific probe that discriminates a normal
or mutant allele. Alternatively, the method can be performed using
an allele-specific primer and a labeled probe that binds to
amplified product. In other embodiments, the probe may not
discriminate between a mutant and normal allele.
[0075] In other embodiments, the presence of a mutant Gnaq allele
can be conveniently determined using conventional or
next-generation DNA sequencing, such as pyrosequencing, or other
known sequencing techniques. Other detection methods include
single-stranded conformational polymorphism or restriction fragment
length polymorphism detection methods and denaturing gradient gel
electrophoresis analysis.
[0076] As indicated above, in some embodiments, levels of Gnaq RNA
are detected. Methods of detecting and/or quantifying the level of
Gnaq gene transcripts (mRNA or cDNA made therefrom) using nucleic
acid hybridization techniques are known to those of skill in the
art. For example, expression levels of Gnaq can also be analyzed by
techniques such as RT-PCR, e.g., using real-time RT-PCR using
allele-specific primers or probes, dot blotting, in situ
hybridization, RNase protection, probing DNA microchip arrays, and
the like.
[0077] Overexpression of Gnaq, either mutated sequences or normal
nucleic acid and/or polypeptide sequences, can be detected, e.g.,
using quantitative sequences known in the art such as those
described herein. Overexpression is determined with reference to a
control, e.g., a normal tissue.
[0078] 2. Detection of GNAQ Protein or Activity
[0079] Altered Gnaq expression and/or activity can also be detected
by detecting Gnaq protein or activity. For example, detection of
Gnaq protein activity or the presence of Gnaq proteins that have a
mutation, can be used for diagnostic, prognostic or treatment
monitoring purposes or in screening assays. In some embodiments,
the level of Gnaq or the presence of a normal or mutant Gnaq
polypeptide in a sample is conveniently determined using
immunological assays. In other embodiments, Gnaq activity can be
used to determine the presence of activating mutation of Gnaq in a
biological sample.
[0080] Antibodies can be used to detect Gnaq or can be assessed in
the methods of the invention for the ability to inhibit Gnaq. The
detection and/or quantification of Gnaq can be accomplished using
any of a number of well recognized immunological binding assays. A
general overview of the applicable technology can be found in
Harlow & Lane, Antibodies: A Laboratory Manual (1988) and
Harlow & Lane, Using Antibodies (1999). Other resources include
see also Methods in Cell Biology: Antibodies in Cell Biology,
volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites
& Ten, eds., 7th ed. 1991, and Current Protocols in Immunology
(Coligan, et al. Eds, John C. Wiley, 1999-present). Immunological
binding assays can use either polyclonal or monoclonal antibodies.
In some embodiments, antibodies that specifically detect mutant
Gnaq molecules are employed.
[0081] Commonly used assays include noncompetitive assays (e.g.,
sandwich assays) and competitive assays. In competitive assays, the
amount of Gnaq present in the sample is measured indirectly by
measuring the amount of a known, added (exogenous) Gnaq displaced
(competed away) from an anti-Gnaq antibody by the unknown Gnaq
present in a sample. Commonly used assay formats include
immunoblots, which are used to detect and quantify the presence of
protein in a sample. Other assay formats include liposome
immunoassays (LIA), which use liposomes designed to bind specific
molecules (e.g., antibodies) and release encapsulated reagents or
markers, which are then detected according to standard techniques
(see Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 (1986)).
[0082] Immunoassays also often use a labeling agent to specifically
bind to and label the complex formed by the antibody and antigen.
The labeling agent may itself be one of the moieties comprising the
antibody/antigen complex. Thus, the labeling agent may be a labeled
Gnaq polypeptide or a labeled anti-Gnaq antibody. Alternatively,
the labeling agent may be a third moiety, such as a secondary
antibody, that specifically binds to the antibody/antigen complex
(a secondary antibody is typically specific to antibodies of the
species from which the first antibody is derived). Other proteins
capable of specifically binding immunoglobulin constant regions,
such as protein A or protein G may also be used as the labeling
agent. The labeling agent can be modified with a detectable moiety,
such as biotin, to which another molecule can specifically bind,
such as streptavidin. A variety of detectable moieties are well
known to those skilled in the art.
[0083] The particular label or detectable group used in the assay
is not a critical aspect of the invention, as long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., DYNABEADS.TM.), fluorescent
compounds (e.g., fluorescein isothiocyanate, Texas red, rhodamine,
fluorescein, and the like), radiolabels, enzymes (e.g., horse
radish peroxidase, alkaline phosphatase and others commonly used in
an ELISA), streptavidin/biotin, and colorimetric labels such as
colloidal gold or colored glass or plastic beads (e.g.,
polystyrene, polypropylene, latex, etc.). Chemiluminescent
compounds may also be used. For a review of various labeling or
signal producing systems that may be used, see U.S. Pat. No.
4,391,904.
[0084] Antibodies to Gnaq are commercially available (e.g., Genesis
Biotech, Inc. Taipei County, Taiwan). In some embodiments,
mutations to Gnaq can be detected using antibodies that
specifically bind a mutant form, thus immunoassays can also be used
to detect mutant Gnaq proteins.
[0085] Gnaq or a fragment thereof, e.g., the portion of the peptide
frequently comprising a sequence mutation, may be used to produce
antibodies specifically reactive with Gnaq. For example, a
recombinant Gnaq or an antigenic fragment thereof, is isolated.
Recombinant protein is the preferred immunogen for the production
of monoclonal or polyclonal antibodies. Alternatively, a synthetic
peptide derived from the sequences disclosed herein and conjugated
to a carrier protein can be used as an immunogen. Naturally
occurring protein may also be used either in pure or impure form.
The product is then used to generate antibodies.
[0086] Methods of producing polyclonal and monoclonal antibodies
that react specifically with Gnaq are known to those of skill in
the art (see, e.g., Coligan; Harlow & Lane, both supra). Such
techniques include antibody preparation by selection of antibodies
from libraries of recombinant antibodies in phage or similar
vectors, as well as preparation of polyclonal and monoclonal
antibodies by immunizing rabbits or mice (see, e.g., Huse et al.,
Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546
(1989)). Such antibodies can be used for diagnostic or prognostic
applications, e.g., in the detection of mutated Gnaq.
[0087] Typically, polyclonal antisera with a titer of 10.sup.4 or
greater are selected and tested for cross reactivity against
non-Gnaq proteins or even other related proteins from other
organisms, using a competitive binding immunoassay. Specific
polyclonal antisera and monoclonal antibodies will usually bind
with a Kd of at least about 0.1 mM, more usually at least about 1
.mu.M, optionally at least about 0.1 .mu.M or better, and
optionally 0.01 .mu.M or better.
[0088] In some embodiments, a Gnaq antibody may be used for
therapeutic applications. For example, in some embodiments, such an
antibody may be used to reduce or eliminate a biological function
of Gnaq. That is, the addition of anti-Gnaq antibodies (either
polyclonal or preferably monoclonal) to a patient with SWS, KTWS or
PWS (i.e., a cell population comprising the Gnaq mutation) may
reduce or eliminate the condition.
[0089] In certain embodiments, the antibodies to the Gnaq proteins
for therapeutic applications are humanized antibodies (e.g.,
Xenerex Biosciences, Mederex, Inc., Abgenix, Inc., Protein Design
Labs, Inc.). Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); Marks et
al., J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al.
and Boemer et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, p. 77 (1985) and Boerner et al., J. Immunol.
147(1):86-95 (1991)). Similarly, human antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, e.g., in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et
al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0090] As appreciated by one of ordinary skill in the art, Gnaq
activity can be detected to evaluate expression levels or for
identifying inhibitors of activity. The activity can be assessed
using a variety of in vitro and in vivo assays, including GTP and
GDP binding activity, GTP-hydrolase activity, or measurement of
phospholipase CP. In some embodiments Gnaq activity can be
evaluated using additional endpoints, such as those associated with
transformation or pigmentation. Such assays are described in
greater detail in the examples and section detailing methods of
identifying additional Gnaq inhibitors. Typically Gnaq activity is
determined by measuring the ability to bind a protein to which it
interacts, e.g., an antibody, ligand, or other protein, such as
signaling molecules.
III. Disease Diagnosis/Prognosis/Monitoring
[0091] Gnaq nucleic acid and polypeptide sequences can be used for
diagnosis, prognosis and/or treatment monitoring of SWS, KTWS or
PWS in a patient. For example, as described above, the sequence,
level, or activity of Gnaq in a sample from a patient can be
determined, wherein an alteration, e.g., a decrease in the level of
expression or activity of Gnaq or a sequence mutation in Gnaq,
indicates a reduction in the severity of the condition.
[0092] The methods of the present invention can be used to
determine the optimal course of treatment in a patient with SWS,
KTWS, or PWS. For example, the presence of a sequence mutation in
Gnaq can indicate that certain therapeutics, such as those that
target Gnaq, phospholipase CP, or downstream pathways regulated by
Gnaq will be beneficial to those patients. Upstream pathways are
also relevant, for example, receptors coupled directly to Gnaq
protein. In addition, a correlation can be readily established
between the presence of a defect or sequence mutation in Gnaq, and
the relative efficacy of one or another anti-SWS, KTWS or PWS
agent. Such analyses can be performed, e.g., retrospectively, i.e.,
by analyzing for a Gnaq defect or sequence mutation in samples
taken previously from patients that have subsequently undergone one
or more types of therapy, e.g., therapies that target G-proteins or
phospholipase CP, or other downstream pathways regulated by Gnaq
and correlating the presence of the defect with the known efficacy
of the treatment.
[0093] Often, such methods will be used in conjunction with
additional diagnostic methods. In other embodiments, a tissue
sample known to contain cells harboring the mutation can be
analyzed for Gnaq defects to determine information about the
condition, e.g., the efficacy of certain treatments, such as
therapeutics that target Gnaq, or downstream pathways regulated by
Gnaq.
[0094] In some embodiments, the analysis of cells/alleles for the
presence of Gnaq defects or sequence mutation can be used to
determine the prognosis of a patient with SWS, KTWS, or PWS or for
determining progression of the condition. A "diagnostic presence"
can be increased levels of Gnaq mRNA or protein and/or activity,
and/or the presence of sequence mutations in Gnaq that alter
function.
[0095] Any biological sample suspected of comprising cells
comprising the mutation can be evaluated to determine progression.
For example, tissues from visceral organs, blood, lymph nodes and
the like can be analyzed for the presence of Gnaq sequence
mutations and or increased levels of Gnaq activity.
IV. GNAQ Inhibitors or Modulators and Screening Methods
Therefor
[0096] In another aspect, the present invention includes methods of
treating patients who have a mutation in Gnaq where the method
comprises administering an inhibitor or Gnaq antagonist. Inhibitors
and Gnaq antagonists are known. For example, non-limiting exemplary
inhibitors suitable for use with the present invention can include
specific and nonspecific inhibitors of PKC and various PKC isoforms
including PKC.mu.. and PKC.epsilon.. Exemplary non-limiting
inhibitors suitable for use with the present invention include
staurosporine, the staurosporine analogue CPG41251, bryostatin-1,
KAI-9803, 7-hydroxystaurosporine, L-threo-dihydrosphingosine
(safingol), the non-selective PKC inhibitor (PKC412), ilmofosine
(BM 41 440), Go6976, which is an indolcarbazole that more
specifically inhibits the classical isoforms of PKC, including
PCK.mu., the PKC-alpha antisense inhibitor LY900003, and the
PKC-beta inhibitors LY333531, LY317615 (Enzastaurin). Non-limiting
exemplary inhibitors of phospholipase C.beta. can include
edelfosine and fluvirusin B[2], which are also suitable for use in
the present invention.
[0097] Other inhibitors include inhibitors such as antibodies,
peptide, nucleic acids and the like. As used herein, a Gnaq
inhibitor can be a molecule that modulates Gnaq nucleic acid
expression and/or Gnaq protein activity, or downstream pathways
regulated by Gnaq.
[0098] Method of screening for modulators of compounds can employ,
for example, cells in which Gnaq is over-expressed or mutated. Such
modulators may be candidate Gnaq GTP hydrolase modulators.
Additional Gnaq inhibitors can be identified by assaying for Gnaq
activity, e.g., GTP binding or GTP hydrolase activity. Such assays
employ known Gnaq sequences or fragments, e.g., the guanine binding
domain of Gnaq, or variants thereof .DELTA.n exemplary human Gnaq
polypeptide sequence that could be used in such assays is provided
in SEQ ID NO:13.
[0099] Activity assays are used to identify inhibitors that can be
used as therapeutic agents, e.g., antibodies to Gnaq and
antagonists of Gnaq activity. Inhibitors of Gnaq activity are
tested using Gnaq polypeptides, either recombinant or naturally
occurring. The protein can be isolated, expressed in a cell,
expressed in tissue or in an animal, either recombinant or
naturally occurring. For example, transformed cells can be used.
Modulation is tested using one of the in vitro or in vivo assays
described herein. Activity can also be examined in vitro with
soluble or solid state reactions, using a Gnaq fragment that binds
to another protein, e.g., phospholipase CP, or GTP.
[0100] In another embodiment, mRNA and/or protein expression levels
can be measured to assess the effects of a test compound on Gnaq
expression levels. A host cell expressing Gnaq is contacted with a
test compound for a sufficient time to effect any interactions, and
then the level of mRNA or protein is measured. The amount of time
to effect such interactions may be empirically determined, such as
by running a time course and measuring the level of expression as a
function of time. The amount of expression may be measured by using
any method known to those of skill in the art to be suitable.
[0101] The amount of expression is then compared to the amount of
expression in the absence of the test compound. A substantially
identical cell may be derived from the same cells from which the
recombinant cell was prepared but which had not been modified by
introduction of heterologous DNA. A difference in the amount of
expression indicates that the test compound has in some manner
altered Gnaq levels.
[0102] In some assays to identify Gnaq inhibitors, samples that are
treated with a potential inhibitor are compared to control samples
to determine the extent of modulation. Control samples without the
mutation and untreated with candidate inhibitors are assigned a
relative activity value of 100. Inhibition of Gnaq is achieved when
the activity value relative to the control is about 80%, about 50%,
or about 25-0%.
[0103] The compounds tested as inhibitors of Gnaq can be any small
chemical compound, or a biological entity, e.g., a macromolecule
such as a protein, sugar, nucleic acid or lipid. Alternatively,
modulators can be genetically altered versions of Gnaq. Typically,
test compounds will be small chemical molecules and peptides or
antibodies.
[0104] In some embodiments, the agents have a molecular weight of
less than 1,500 daltons, and in some cases less than 1,000, 800,
600, 500, or 400 daltons. The relatively small size of the agents
can be desirable because smaller molecules have a higher likelihood
of having physiochemical properties compatible with good
pharmacokinetic characteristics, including oral absorption than
agents with higher molecular weight. For example, agents less
likely to be successful as drugs based on permeability and
solubility were described by Lipinski et al. as follows: having
more than 5 H-bond donors (expressed as the sum of OHs and NHs);
having a molecular weight over 500; having a Log P over 5 (or M Log
P over 4.15); and/or having more than 10 H-bond acceptors
(expressed as the sum of Ns and Os). See, e.g., Lipinski et al. Adv
Drug Delivery Res 23:3-25 (1997). Compound classes that are
substrates for biological transporters are typically exceptions to
the rule.
[0105] Essentially any chemical compound can be used as a potential
modulator or ligand in the assays of the invention. Most often,
compounds can be dissolved in aqueous or organic (especially
DMSO-based) solutions. The assays are designed to screen large
chemical libraries by automating the assay steps, which are
typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland)
and the like.
[0106] Certain screening methods involve screening for a compound
that modulates the expression of Gnaq. Such methods generally
involve conducting cell-based assays in which test compounds are
contacted with one or more cells expressing Gnaq and then detecting
a decrease in expression (either transcript or translation
product).
[0107] Expression can be detected in a number of different ways. As
described herein, the expression levels of the protein in a cell
can be determined by probing the mRNA expressed in a cell with a
probe that specifically hybridizes with a Gnaq transcript (or
complementary nucleic acid derived therefrom). Alternatively,
protein can be detected using immunological methods in which a cell
lysate is probed with antibodies that specifically bind to the
protein.
[0108] Other cell-based assays are reporter assays conducted with
cells that do not express the protein. Often, these assays are
conducted with a heterologous nucleic acid construct that includes
a promoter that is operably linked to a reporter gene that encodes
a detectable product.
V. Treatment and Administration of Pharmaceutical Compositions
[0109] Inhibitors of Gnaq can be administered to a patient for the
treatment of SWS, KTWS or PWS having a sequence mutation in Gnaq.
As described in detail below, the inhibitors are administered in
any suitable manner, optionally with pharmaceutically acceptable
carriers. In some embodiments, inhibitors of PKC or phospholipase
C.beta. are administered. Protocols for the administration of
inhibitors are known and can be further optimized for SWS, KTWS or
PWS patients based on principles known in the pharmacological arts
(Remington's Pharmaceutical Sciences, 17th ed., 1989).
[0110] The inhibitors can be administered to a patient at
therapeutically effective doses to prevent, treat, or control the
condition. The compounds are administered to a patient in an amount
sufficient to elicit an effective protective or therapeutic
response in the patient. An effective therapeutic response is a
response that at least partially arrests or slows the symptoms or
complications of the disease. An amount adequate to accomplish this
is defined as "therapeutically effective dose." The dose will be
determined by the efficacy of the particular Gnaq inhibitor
employed and the condition of the subject, as well as the body
weight or surface area of the area to be treated. The size of the
dose also will be determined by the existence, nature, and extent
of any adverse effects that accompany the administration of a
particular compound in a particular subject.
[0111] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, for example, by determining the LD.sub.50
(the dose lethal to 50% of the population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and can be expressed as the ratio, LD.sub.50/ED.sub.50.
Compounds that exhibit large therapeutic indices are preferred.
While compounds that exhibit toxic side effects can be used, care
should be taken to design a delivery system that targets such
compounds to the site of affected tissue to minimize potential
damage to normal cells and, thereby, reduce side effects.
[0112] The data obtained from cell culture assays and animal
studies can be used to formulate a dosage range for use in humans.
The dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage can vary within this range depending
upon the dosage form employed and the route of administration. For
any compound used in the methods of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (the concentration of the test compound that achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma can be measured, for example, by
high performance liquid chromatography (HPLC). In general, the dose
equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a
typical subject.
[0113] Pharmaceutical compositions for use in the present invention
can be formulated by standard techniques using one or more
physiologically acceptable carriers or excipients. The compounds
and their physiologically acceptable salts and solvates can be
formulated for administration by any suitable route, including via
inhalation, topically, nasally, orally, parenterally (e.g.,
intravenously, intraperitoneally, intravesically or intrathecally)
or rectally.
[0114] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients,
including binding agents, for example, pregelatinised maize starch,
polyvinylpyrrolidone, or hydroxypropyl methylcellulose; fillers,
for example, lactose, microcrystalline cellulose, or calcium
hydrogen phosphate; lubricants, for example, magnesium stearate,
talc, or silica; disintegrants, for example, potato starch or
sodium starch glycolate; or wetting agents, for example, sodium
lauryl sulphate. Tablets can be coated by methods well known in the
art. Liquid preparations for oral administration can take the form
of, for example, solutions, syrups, or suspensions, or they can be
presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations can be
prepared by conventional means with pharmaceutically acceptable
additives, for example, suspending agents, for example, sorbitol
syrup, cellulose derivatives, or hydrogenated edible fats;
emulsifying agents, for example, lecithin or acacia; non-aqueous
vehicles, for example, almond oil, oily esters, ethyl alcohol, or
fractionated vegetable oils; and preservatives, for example, methyl
or propyl-p-hydroxybenzoates or sorbic acid. The preparations can
also contain buffer salts, flavoring, coloring, and/or sweetening
agents as appropriate. If desired, preparations for oral
administration can be suitably formulated to give controlled
release of the active compound.
[0115] For administration by inhalation, the compounds may be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant, for example, dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide,
or other suitable gas. In the case of a pressurized aerosol, the
dosage unit can be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of, for example, gelatin
for use in an inhaler or insufflator can be formulated containing a
powder mix of the compound and a suitable powder base, for example,
lactose or starch.
[0116] The compounds can be formulated for parenteral
administration by injection, for example, by bolus injection or
continuous infusion. Formulations for injection can be presented in
unit dosage form, for example, in ampoules or in multi-dose
containers, with an added preservative. The compositions can take
such forms as suspensions, solutions, or emulsions in oily or
aqueous vehicles, and can contain formulatory agents, for example,
suspending, stabilizing, and/or dispersing agents. Alternatively,
the active ingredient can be in powder form for constitution with a
suitable vehicle, for example, sterile pyrogen-free water, before
use.
[0117] The compounds can also be formulated in rectal compositions,
for example, suppositories or retention enemas, for example,
containing conventional suppository bases, for example, cocoa
butter or other glycerides.
[0118] Furthermore, the compounds can be formulated as a depot
preparation. Such long-acting formulations can be administered by
implantation (for example, subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the compounds can be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0119] The compositions can, if desired, be presented in a pack or
dispenser device that can contain one or more unit dosage forms
containing the active ingredient. The pack can, for example,
comprise metal or plastic foil, for example, a blister pack. The
pack or dispenser device can be accompanied by instructions for
administration.
V. Kits
[0120] The invention also provides kits for diagnostic, prognostic
or therapeutic applications. For diagnostic/prognostic
applications, such kits may include any or all of the following:
assay reagents, buffers, Gnaq probes, primers, antibodies, or the
like.
[0121] In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the practice of the
methods of this invention. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. Such media may include
addresses to internet sites that provide such instructional
materials.
[0122] Without further elaboration, it is believed that one skilled
in the art, using the preceding description, can utilize the
present invention to the fullest extent. The following examples are
illustrative only, and not limiting of the remainder of the
disclosure in any way whatsoever.
Examples
[0123] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices,
and/or methods described and claimed herein are made and evaluated,
and are intended to be purely illustrative and are not intended to
limit the scope of what the inventors regard as their invention.
Efforts have been made to ensure accuracy with respect to numbers
(e.g., amounts, temperature, etc.) but some errors and deviations
should be accounted for herein. Unless indicated otherwise, parts
are parts by weight, temperature is in degrees Celsius or is at
ambient temperature, and pressure is at or near atmospheric. There
are numerous variations and combinations of reaction conditions,
e.g., component concentrations, desired solvents, solvent mixtures,
temperatures, pressures and other reaction ranges and conditions
that can be used to optimize the product purity and yield obtained
from the described process. Only reasonable and routine
experimentation will be required to optimize such process
conditions.
Materials and Methods
[0124] Informed Consent. All studies were performed with approval
of the Johns Hopkins or Duke University Institutional Review Board.
De-identified samples were obtained with informed consent from
subjects with SWS or from the NICHD Maryland Brain and Tissue Bank
for Developmental Disorders (MBTBDD). Age, sex, ethnicity and
syndrome status (apparently non-syndromic PWS or SWS) were
confirmed and recorded from the source along with information
regarding surgery, autopsy or procedure providing the tissue.
[0125] Whole Genome Sequencing. Genomic DNA was purified from
affected or unaffected blood or tissue from three individuals (n=6)
using a Qiagen Puregene (blood samples) or DNAeasy (skin and brain)
extraction kit and quantitated using a SybrGreen assay. Whole
genome sequencing was performed on an Illumina HiSeq 2000 at
Illumina, Inc. (San Diego, Calif.) to 33.3.times. to 50.8.times.
mean depth of coverage. Paired end reads were aligned to the hg19
human reference using the Illumina Genome Analyzer Pipeline.
Somatic single nucleotide variants (SNV) or insertion/deletions
(indel) were detected using Strelka. Post-call filtration was
applied using the author's recommended parameters to remove
spurious SNVs and indels resulting from homo-polymer repeats and
abnormally high read depth regions. Somatic SNV calls from Strelka
(from autosomes as well as X chromosome) to the affected samples
were identified, and mutant allele frequencies were calculated for
each affected/normal pair of WGS. These were identified as
potential somatic SNVs. There SNV cells and ranked in order of
predicted functional effect using VAAST. 1000 Genomes variants were
specified as background to remove variants found in a normal
population, and RefSeq genes were used as regions of interest.
[0126] Targeted Amplicon Re-Sequencing. An amplicon consisting of
168 bp from GNAQ exon 4 and adjacent intronic sequence
(NC_000009.11 80,412,463-80,412,630), encompassing the GNAQ c.548G
residue (NC_002072.3), was PCR amplified in a two-stage reaction.
For each sample a unique DNA barcode, sequencing adapters, and
Illumina flow-cell clustering adapters were added. Barcode
sequences were generated using a 7 nucleotide Hamming encoding
scheme, allowing for correction of a single base miscall. Paired
end sequencing of barcoded amplicons was performed using an
Illumina MiSeq, producing 151 nt paired end reads. Reads were
aligned to the hg1 human reference using BWA 0.6.2 with base
quality trimming Q30. Samtools mpileup of base calls greater than
Q30 was used to determine allele counts at the mutation site. Bpipe
pipelines not shown. Aligned exome reads from the 1000 Genomes
Project{Consortium:2011gj} (alignment release 2011-11-14) were
evaluated at the c.548 position for base calls supporting the
c.548G>A mutation. Only exomes having a read depth of greater
than 100 at the variant site were evaluated. Samples from both
amplicon sequencing and 1000 Genomes Project exomes were determined
to have a mutation if the percent of reads supporting a mutation
exceeded 10 times (1%) the expected base miscall rate (0.1%). The
median read depth for amplicon sequencing was selected on the basis
of 1,000,000 simulations of a random draw from an allele pool with
1% mutant allele frequency.
[0127] Plasmids. Full length wild type GNAQ plasmid was purchased
from Origene (Rockville, Md.). This TruORF Gold clone contains the
entire reading frame plus Myc and flag epitope tags at the carboxyl
terminus. Specific mutations, c.548G>A, p.Arg183Gln, and
626A>T, p.Gln209Leu, were introduced into the clone using
primers for site directed mutagenesis. Clones were sequenced to
verify that no other changes were present. pSRE (serum response
element)-Luc (Agilent Technologies) and pSV40-RL (Roche) were used
as reporter plasmids for the luciferase assay.
[0128] Luciferase Assay. GNAQ-WT, -R183Q, or -Q209L, pSRE-Luc and
pSV40-RL at a ratio of 5:200:1 ng were transfected into 293T cells
using Fugene 6 (Promega). Cells were lysed after 20-24 hours
incubation and the luciferase activity was measured using the
Dual-Luciferase Reporter Assay System (Promega) on a Polarstar
Optima plate reader (BMG Labtech, Germany). Experiments were
performed in triplicate.
[0129] SNaPshot Assay. DNA was extracted from archived,
formalin-fixed, paraffin-embedded port-wine stain tissue samples
according to the manufacturer's protocol (Gentra PureGene, Qiagen).
Primers for exon 4 (Table 1) were used to amplify genomic DNA from
each of the samples and electrophoresed on a 1% agarose gel. PCR
products were extracted using GeneClean Turbo (MP Bio). Purified
PCR products were interrogated for their sequence at position c.548
using the SNaPshot Multiplex Kit (Life Technologies, Grand Island,
N.Y.) and analyzed on an ABI Prism 3130. Reference and mutant
allele frequencies were calculated based on the area of the
resulting peaks.
TABLE-US-00001 TABLE 1 SNaPshot Primer Sequences GNAQ exon 4
amplicon Primer sequence Forward 5' ATTGTGTCTTCCCTCCTCTA (SEQ ID
NO: 1) Reverse 5' GGTTTCATGGACTCAGTTAC (SEQ ID NO: 2) SNaPshot
Forward 5' CGCAACAAGATGTGCTTAGAGTTC (SEQ ID NO: 3) SnaPshot Reverse
5' TCCCTGTGGTGGGGACT (SEQ ID NO: 4) GNAQ exon 4 amplicon forward
and reverse primers produce a 207 bp amplicon comprising the
sequence of interest. Primers for single base pair extension
(SNaPshot) are designed immediately upstream and downstream of the
base of interest, c.548G > A.
TABLE-US-00002 TABLE 2 SNaPshot Primer Sequences. GNA11 exon 4
amplicon Primer sequence Forward 5' GAGCACCCACCGCTGTGTTG (SEQ ID
NO: 5) Reverse 5' GGCAAATGAGCCTCTCAGTGC (SEQ ID NO: 6) SnaPshot
Forward 1 5' CAGGACGTGCTGCGGGTC (547C > T) (SEQ ID NO: 7)
SnaPshot Reverse 1 5' TGCCGGTGGTGGGCACGC (547C > T) (SEQ ID NO:
8) SnaPshot Forward 2 5' GCAGGACGTGCTGCGGGT (546C > T) (SEQ ID
NO: 9) SnaPshot Forward 3 5' AGGACGTGCTGCGGGTCC (548G > A) (SEQ
ID NO: 10) SnaPshot Reverse 3 5' ATGCCGGTGGTGGGCACG (548G > A)
(SEQ ID NO: 11) GNA11 exon 4 amplicon forward and reverse primers
produce a 207 bp amplicon comprising the sequences of interest.
Primers for single base pair extension (SNaPshot) are designed
immediately upstream and downstream of the bases of interest,
c.547C > T, c.546C > T, and c.548G > A.
[0130] Cell Culture and Western Blotting. Human embryonic kidney
HEK293T (293T) cells (ATCC, Manassas, Va.) were maintained in
Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10%
fetal bovine serum at 37.degree. C. in 5% CO.sub.2. Cells were
grown on 100 mm.sup.2 plates and transfected with 6 .mu.g plasmid
DNA using FuGene 6 reagent (Roche, Indianapolis, Ind.) according to
the manufacturer's protocol. Cell lysates were analyzed by western
blotting using standard methods. Antibodies recognizing p44/42 MAPK
(Erk1/2) (#9102), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204,
#9101), p38 MAPK (#9212), Phospho-p38 MAPK (Thr180/Tyr182, #9211),
Akt (#9272), Phospho-Akt (Ser473, #4058), MARCKS (#5607),
Phospho-MARCKS (Ser167/170 #8722) and Phospho-JNK (Thr183/Tyr185,
#9251) were obtained from Cell Signaling Technology. JNK (SC-474)
antibodies were obtained from Santa Cruz. Mouse anti-Flag M2
(1:1000, Sigma) and Mouse anti-alpha tubulin (1:500, DSHB, U. of
Iowa) were also used. Protein bands were visualized using secondary
antibodies conjugated to HRP (1:3000, BioRad) followed by
incubation with Pierce SuperSignal West Pico Chemiluminescent
Substrate (Thermo Fisher Scientific, Rockford, Ill.) and analyzed
using the G:BOX gel documentation system (Syngene).
Tetradecanoylphorbol acetate (TPA #4174) was obtained from Cell
Signaling Technology, and used at 200 nM for 20-30 mins.
Results
[0131] Example 1: Identification of GNAQ Somatic Variant. To test
the hypothesis that SWS is associated with a somatic mosaic
mutation, we sequenced the whole genomes of paired DNA samples from
affected regions (biopsied PWS or hemispherectomized brain tissue)
and matched, presumed normal regions (blood, skin, or brain) from
three individuals with SWS. This resulted in the identification of
1,294 somatic SNVs found in at least one of three affected samples.
We calculated the prevalence of the variant allele at each of these
1,294 sites for all affected and normal samples and identified SNVs
shared between two and three affected samples (n=658), and not
present in any normal samples. We functionally annotated and ranked
the 1,294 somatic SNVs using VAAST. This resulted in the
identification of a single non-synonymous candidate SNV supported
by reads present in all three affected samples and not the presumed
normal samples, a c.548G>A nucleotide transition in GNAQ on
chromosome 9q21, encoding guanine nucleotide binding protein (G
protein), q polypeptide. The variant is predicted to result in the
amino-acid substitution p.Arg183Gln. The affected arginine residue,
at position 183, is conserved in 24 human genes paralogous to
G.alpha..
[0132] Example 2: Detection of GNAQ c.548G>A Somatic Variant in
SWS Samples. A collection of tissues composed of PWS and visibly
normal skin from subjects with SWS (n=23 samples from 10 subjects),
apparently non-syndromic PWS and visibly normal skin from subjects
without SWS (n=14 samples from 13 subjects), brain from individuals
with SWS (n=50 from 18 subjects), or control brain from presumably
normal individuals (n=6 from 6 subjects) was obtained from research
subjects or from the NICHD Brain and Tissue Bank for Developmental
Disorders. Amplicon sequencing and single-base extension
interrogation (SNaPshot analysis) were used to interrogate each
tissue sample for the c.548G>A mutation. SNaPshot specificity
was assayed by testing five normal brain controls (data not shown).
For subjects having biological or technical replicates, we call the
subject positive for the mutation if at least one tissue sample
tested positive (>=1% mutant allele), and negative if every
tissue sample tested negative for the mutation (<1% mutant
allele). 100% (9/9) of subjects with SWS were positive for the
c.548G>A mutation in PWS skin tissue. 85.7% (6/7) of subjects
with SWS were negative in visibly normal skin samples. 92.3%
(12/13) of subjects with apparently non-syndromic PWS were positive
for the mutation in PWS skin samples (Table 3). 83.3% (15/18) of
subjects with SWS were positive for the c.548G>A mutation in
brain samples. 100% of normal brain samples from 6/6 subjects from
the control population were negative. 100% (4/4) of formalin-fixed,
paraffin-embedded CCM brain samples from subjects with cerebral
cavernous malformation (CCM) were negative (Table 4). 99.3%
(664/669) of exomes from the 1000 Genomes database were negative.
For amplicon sequencing, mutant allele frequencies ranged from 1 to
18.1%, and read depth ranged from 2,446 to 93,008, median 12,947.
1000 Genomes exome mutant allele frequencies ranged from 1 to 1.5%,
and read depth ranged from 100 to 453, median 271. GNA11 mutations
have also been found in uveal melanoma. We tested GNAQ Arg183Gln
mutation-negative SWS and PWS samples for the presence of
previously identified GNA11 mutations (p.Arg183Cys, c.547C>T and
c.546C>T; p.Arg183His, c.548G>A; p.Gln209Leu, c.626A>T and
c.627G>A; p.Gln209Pro, c.626A>C) using SNaPshot analysis. We
did not detect any of these mutations (data not shown).
TABLE-US-00003 TABLE 3 Summary of somatic mutation of GNAQ in skin
samples. Mutant Allele ID Mutation PWS SWS Frequency (%) Samples 1
Y Y Y 3.60 1 1 N N Y 0.11 1 2 Y Y Y 3.17 1 2 N N Y 0.13 1 3 Y Y Y
6.06-6.46 2 3 N N Y 0.62-0.93 2 4 Y Y Y 3.50-4.51 2 4 N N Y
0.13-0.90 2 5 Y Y Y 3.38 1 5 N N Y 0.11 1 6 Y Y Y 3.99 1 7 Y Y Y
2.00-2.16 3 7 N N Y 0.09 1 8 Y Y Y 4.08 1 8 N N Y 0.06 1 9 Y Y N
5.58 1 10 Y Y N 2.76 1 10 Y N N 1.14 1 11 Y Y N 6.70 1 12 N Y N
0.00 1 13 Y Y N 5.90 1 14 Y Y N 6.20 1 15 Y Y N 14.20 1 16 Y Y N
1.70 1 17 Y Y N 4.50 1 18 Y Y N 5.30 1 19 Y Y N 4.70 1 20 Y Y N
4.30 1 21 Y Y N 18.10 1 22 Y Y Y 5.00 1
[0133] Data are shown for c.548G>A which results in the
p.Arg183Gln amino acid substitution. This position corresponds to
position 80,412,493 on chromosome 9 (NCBIv37). Abbreviations: ID,
individual identifier; Mutation denotes whether the p.Arg183Gln
substitution is present (i.e., the mutant allele frequency is above
1%); PWS, port-wine stain; SWS, Sturge-Weber syndrome; % Mutant
Allele, percent mutant allele divided by total alleles (ranges for
representations of multiple samples); Samples, number of replicate
samples assayed. Samples from non-syndromic PWS (PWS-Y, SWS-N), PWS
from an individual with SWS (PWS-Y, SWS-Y), or normal tissue from
an individual with SWS (PWS-N, SWS-Y). The p.Arg183Gln substitution
was considered to be present if the mutant allele frequency was
more than 1%. The mutant allele frequency was calculated as the
percentage of mutant alleles divided by total alleles (with ranges
shown in the case of multiple samples).
TABLE-US-00004 TABLE 4 Summary of somatic mutation of GNAQ in brain
samples. ID Mutation SWS % Mutant Allele Samples 7 Y Y 5.57-5.63 2
23 Y Y 5.56-5.78 2 24 Y Y 2.67-3.51 2 25 N Y 0.02-0.10 2 26 Y Y
0.13-3.06 4 27 Y Y 2.19-5.12 2 28 Y Y 6.95-8.13 4 29 Y Y 6.04-11.15
5 30 Y Y 4.14 1 31 Y Y 4.78 1 32 Y Y 0.22-1.48 4 33 Y Y 4.04-5.74 2
34 N Y 0.05-0.12 2 35 Y Y 0.05-1.51 7 36 Y Y 0.35-6.03 5 37 Y Y
5.74-6.49 2 38 N Y 0.03-0.05 2 39 Y Y 1.83 1 40 N N 0.11 1 41 N N
0.05 1 42 N N 0.08 1 43 N N 0.09 1 44 N N 0.04 1 45 N N 0.04 1 46 N
CCM 0.00 1 47 N CCM 0.00 1 48 N CCM 0.00 1 49 N CCM 0.00 1 Data are
shown for c.548G > A which results in the p.Arg183Gln amino acid
substitution. Abbreviations are as described in Table 1. CCM =
cerebral cavernous malformation. Samples were from individuals with
SWS (SWS-Y), normal controls (SWS-N) or with CCM.
[0134] Example 3: Effect of Mutation on MAPK Signaling Pathway. The
somatic amino-acid substitutions GNAQ p.Gln209Leu and GNAQ
p.Arg183Gln are found in uveal melanoma. The more common
p.Gln209Leu has been shown to hyper-activate the MAP kinase
pathway. We therefore examined whether p.Arg183Gln would likewise
hyper-activate the MAP kinase pathway. As shown in FIG. 2A, when
compared with cells transfected with non-mutant GNAQ, both
p.Gln209Leu and p.Arg183Gln induced a significant activation of ERK
(p<0.05). However, the activation induced by p.Arg183Gln was
modest compared to p.Gln209Leu. We also examined the effect of
these substitutions on additional downstream signaling pathways.
Neither substitution showed an effect on the AKT signaling pathway.
p.Gln209Leu strongly activated p38 and JNK, other MAPK pathway
members, while p.Arg183Gln did not (FIG. 2B,C). These data show
that p.Arg183Gln represents a gain-of-function that activates
downstream signaling pathways. However, when compared to the common
substitution p.Gln209Leu found in uveal melanoma tissue, the effect
of p.Arg183Gln in MAPK signal transduction appeared to be both
weaker and less promiscuous in its activation of downstream
effectors.
[0135] Example 4: Effect of GNAQ Mutation on SRE Promoter Activity.
A different substitution in GNAQ at the same amino acid residue,
p.Arg183Cys, had previously been shown to hyper-stimulate the serum
response element (SRE) in a promoter reporter assay. We
investigated whether the p.Arg183Gln substitution had the same
stimulatory effect on SRE promoter activity. We transfected 293T
cells with pSRE-Luc, pSV40-RL (reporter constructs) and either
GNAQ, GNAQ p.Arg183Gln, or GNAQ p.Gln209Leuplasmids, and measured
luciferase activity after 24 hours. Both p.Gln209Leu and
p.Arg183Gln showed significantly increased reporter activity over
non-mutant GNAQ (p<0.05), confirming that the p.Arg183Gln
mutation is a gain-of-function/activating mutation (FIG. 2F). In
this assay, the p.Gln209Leu substitution again showed a stronger
effect than p.Arg183Gln.
DISCUSSION
[0136] Rudolf Happle first suggested that sporadic asymmetric
and/or scattered birth defects involving the skin are caused by
somatic mosaic mutations that would be lethal if occurring in very
early embryonic development. Somatic mosaic activating mutations
have been identified in several disorders including the
McCune-Albright syndrome and the Proteus syndrome. In the present
study, we report that a specific somatic mosaic activating mutation
in GNAQ is associated with both the neurocutaneous disorder
Sturge-Weber syndrome and apparently non-syndromic port-wine
stains. GNAQ encodes a member of the q class of G-protein alpha
subunits mediating signals between G-protein-coupled receptors
(GPCRs) and downstream effectors. We have identified somatic mosaic
GNAQ p.Arg183Gln amino acid substitutions in both SWS and PWS, and
have shown that this mutation, much like GNAQ p.Gln209Leu,
activates downstream MAP kinase signaling. GNAQ Arg183 is conserved
in the GTP binding pocket of all human G.alpha. subunits, where it
plays a critical role in the hydrolysis of GTP, the key step
required for inactivation of the protein. Substitution of cysteine
at this position leads to a reduction in the intrinsic GTPase
activity, leading to increased signaling activity.
[0137] Activating mutations in genes encoding G.alpha. subunits
have been previously associated with relevant phenotypes including
McCune-Albright syndrome, which presents with skeletal
abnormalities and abnormal skin pigmentation. Activating somatic
GNAQ mutations have been identified in blue nevi and the more
extensive nevus of Ota. The appearance of these melanocytic naevi,
when co-localized with PWS, is termed phakomatosis
pigmentovascularis (PPV), and is occasionally found in association
with SWS. Mutations in GNAQ were also identified in a chemical
mutagenesis screen for a dark-skin phenotype in laboratory mice.
Two of the dark-skin mutant alleles were identified at positions
corresponding to human GNAQ p.Val179Met and p.Phe335Leu. These
germline amino acid substitutions cause an increase in the number
of neural crest cells that differentiate into melanoblasts. The
abnormal early melanocytic development resulting from these
mutations in the neural crest cells is mediated through the G
protein-coupled receptor (GPCR) endothelin. Endothelin also has
important roles in vasculogenesis, and therefore dysregulation of
this GPCR secondary to the GNAQ p.Arg183Gln mutation in SWS and
non-syndromic PWS may also bring about vascular malformation.
[0138] A somatic activating mutation may have oncogenic potential.
In fact, somatic mutations of GNAQ in melanocytes are associated
with uveal melanoma. The most common mutation, GNAQ p.Gln209Leu, is
an activating mutation that leads to increased downstream signaling
through the MAP kinase pathway. The activation of this pathway
increases cell proliferation and inhibits apoptosis. A few uveal
melanomas have been reported to harbor somatic GNAQ p.Arg183Gln,
although the functional consequence of this substitution has not
been reported. The pathogenesis of uveal melanoma is likely to be
very different from non-syndromic port-wine stains and Sturge-Weber
syndrome. Melanomas are frequently found to have several somatic
mutations. We find no evidence of accumulating mutations in our
three paired whole genome sequences from affected and unaffected
tissue from subjects with SWS. In addition, SWS, PWS, and
melanocytic naevi are thought to originate during fetal
development; therefore the effects of the same GNAQ somatic
mutation may be quite different depending on the cell type and the
point in development at which they arise. There are reported cases
of uveal melanoma associated with PPV, and it is possible that the
coincidence of the blue nevus and PWS phenotype in a SWS patient
may indicate an increased risk for onset of uveal melanoma,
although such coincidences are rare.
[0139] We have shown that the G.alpha..sub.q p.Arg183Gln
substitution can activate ERK and does not activate p38 or JNK in
the same fashion as p.Gln209Leu. We propose that the moderate
activation of ERK and/or differential effect on p38 and JNK
pathways may contribute to the PWS and syndromic characteristics of
SWS. This may occur through either upstream regulation of
G.alpha..sub.q or downstream modulation of the GPCR mediated
signaling cascade. To provide insight into possible mechanisms
underlying the partial activation of G.alpha. downstream signaling,
we consider an interesting corollary in other G.alpha. proteins.
The RGS (Regulator of G protein signaling) proteins serve as
GTPase-activating proteins (GAPs) for G.alpha. proteins, inhibiting
downstream activation. Of these, RGS4 regulates G.alpha..sub.q and
G.alpha..sub.i, while RGS2 is selective for G.alpha..sub.q. Upon
examination of the ability of RGS4 to regulate G.alpha..sub.i1 with
activating mutations in positions p.Arg178Cys and p.Gln204Leu,
homologous to G.alpha..sub.q p.Arg183Gln and p.Gln209Leu, it was
found that all regulatory ability was lost for p.Gln204Leu, while
GTPase activity was partially maintained for p.Arg178Cys. Thus the
weaker and less promiscuously activating effects of GNAQ
p.Arg183Gln, when compared to GNAQ p.Gln209Leu, may be a result of
partial regulation by a member of the RGS family. G.alpha.q is also
able to initiate sustained RhoA and Rac1 activation independently
of PLC-.beta., via direct interaction with Trio, a guanine
nucleotide exchange factor (GEF). It was shown that
G.alpha..sub.q-mediated oncogenic proliferation, mediated through
p38 and JNK, was reduced significantly following Trio knockdown
without affecting PLC or ERK activation levels. This provides a
possible mechanism, related to altered affinity of protein-protein
interactions with both regulators (RGS family) and cascade
activators (Trio), to explain the non-oncogenic proliferation seen
in SWS/PWS. We hypothesize that only the weaker effect of somatic
GNAQ p.Arg183Gln would be compatible with the abnormal but
non-lethal development of the cerebrovascular system seen in SWS.
We also hypothesize that during embryonically vulnerable periods,
moderately increased baseline signaling downstream of
G.alpha..sub.q, and/or dysregulated signaling via GPCRs such as
endothelin, may result in malformed, progressively dilated, and
abnormally innervated PWS blood vessels. There is some evidence in
the literature to support this hypothesis. Shirazi et al. reported
the localization of phosphorylated ribosomal protein S6 (RPS6),
which is downstream of MAPK signaling, to endothelial cells lining
the lumenal wall of abnormal blood vessels in PWS tissue from
patients with SWS.
[0140] The non-syndromic PWS may represent a late origin of the
somatic GNAQ mutation in a vascular endothelial cell, whereas the
SWS mutation may occur earlier in development in a progenitor cell
that is a precursor to a larger variety of cell types and tissues,
leading to the syndromic phenotype. Mutation during early
development might lead to SWS, whereas the
TABLE-US-00005 TABLE 5 Mutation Positive Forward Reverse Mutant
Mutant Samples Allele % Allele % Type of Sample Other 2P 2.90 4.84
PWS, SWS Same as 33P * 8P 0.74 11.01 PWS 8F 4.36 40.94 PWS 20P 3.67
5.02 PWS, SWS Same as 11P* 33P 4.31 5.28 PWS, SWS Same as 2P* but
different specimen Legend: IH--Infantile Hemangioma; PWS--port wine
stains; SWS--Sturge Weber Syndrome; CH--Congenital Hemangioma;
PS--Proteus Syndrome; LM--Lmphatic Malformation; HFM--hamartomatous
fibroadipose hypertrophy; MS--mafucci syndrome; Sch--spindle cell
hemangioma; dermal dendritic melanosis; *Different specimen
same mutation arising later in development might lead to the
isolated, non-syndromic PWS. We observe that 0.7% (5/669) of
samples of blood from the 1000 Genomes database assayed for the
GNAQ p.Arg183Gln mutation were positive. The reported prevalence of
PWS is 0.3-0.5%. We hypothesize therefore that the 0.7% prevalence
in this database represents the occurrence of PWS in this
population.
[0141] Our data unify the underlying mechanism of SWS and PWS and
add a molecular basis to a decades-old hypothesis on the etiology
of these malformations. The scientific and translational novelty of
this discovery lies in associating both apparently non-syndromic
port-wine stains and Sturge-Weber syndrome with a mutation in a
specific gene, a specific genetic mechanism and a set of potential
pathways, thereby providing foundation for further scientific and
clinical research.
TABLE-US-00006 TABLE 6 Mutation Negative Sample Type of Sample
Other 1P PTEN mutation Associated w/ vascular anomaly 3P/3F KTS
4P/4F Capillary malformation Not PWS 5P AVM w/ Same as 15P*
reactive capillary proliferation 6P/6F Vascular malformation rare
phakomatosis vascularis associated w/ ddm 9F PWS 10P/10F
Non-Involuting CH 12F IH 13F LM, possible PS 23F* 14F PS Hfh w/
overlying epidermal nevus 15P AVM Same as 5P* 16P Dermal melanosis
19F KTS 21F KTS 22F PWS 23F LM, PS Same 13P and F* 25P AVM 27F KTS
29F KTS 31P KTS 32P Sch in patient with MS 34P Segmental IH 7P
Angiokeratoma 11P Normal esophageal tissue Same as 20P* with SWS
12P IH 13P LM Proteus Syndrome 17P KTS 18P KTS
[0142] Above is data from a large number of vascular anamolies
which were tested in a blinded fashion for the mutation in GNAQ.
From these two tables can be seen that only the Sturge-Weber
syndrome and port-wine birthmark tissue samples were positive for
the GNAQ mutation. This data provides additional support for the
specificity of the mutation causing SWS and PWB; this mutation is
not linked with the other numerous vascular malformations and
anamolies listed above.
Sequence CWU 1
1
19120DNAArtificial SequenceGNAQ exon 4 amplicon forward primer
1attgtgtctt ccctcctcta 20220DNAArtificial SequenceGNAW exon 4
amplicon reverse primer 2ggtttcatgg actcagttac 20324DNAArtificial
SequenceSnapshot forward primer for c.548G>A 3cgcaacaaga
tgtgcttaga gttc 24417DNAArtificial SequenceSnapshot reverse primer
for c.548G>A 4tccctgtggt ggggact 17520DNAArtificial
SequenceGNA11 exon 4 amplicon forward primer 5gagcacccac cgctgtgttg
20621DNAArtificial SequenceGNA11 exon 4 amplicon reverse primer
6ggcaaatgag cctctcagtg c 21718DNAArtificial SequenceSnapshot
forward primer for c.547C>T 7caggacgtgc tgcgggtc
18818DNAArtificial SequenceSnapshot reverse primer for c.547C>T
8tgccggtggt gggcacgc 18918DNAArtificial SequenceSnapshot forward
primer for c.546C>T 9gcaggacgtg ctgcgggt 181018DNAArtificial
SequenceSnapshot forward primer for c.548G>A 10aggacgtgct
gcgggtcc 181118DNAArtificial SequenceSnapshot reverse primer for
c.548G>A 11atgccggtgg tgggcacg 18122215DNAHomo sapiens
12gggagggtgt gtgtgcgcgc tgtgagcagg gggtgccggc ggggctgcag cggaggcact
60ttggaagaat gactctggag tccatcatgg cgtgctgcct gagcgaggag gccaaggaag
120cccggcggat caacgacgag atcgagcggc agctccgcag ggacaagcgg
gacgcccgcc 180gggagctcaa gctgctgctg ctcgggacag gagagagtgg
caagagtacg tttatcaagc 240agatgagaat catccatggg tcaggatact
ctgatgaaga taaaaggggc ttcaccaagc 300tggtgtatca gaacatcttc
acggccatgc aggccatgat cagagccatg gacacactca 360agatcccata
caagtatgag cacaataagg ctcatgcaca attagttcga gaagttgatg
420tggagaaggt gtctgctttt gagaatccat atgtagatgc aataaagagt
ttatggaatg 480atcctggaat ccaggaatgc tatgatagac gacgagaata
tcaattatct gactctacca 540aatactatct taatgacttg gaccgcgtag
ctgaccctgc ctacctgcct acgcaacaag 600atgtgcttag agttcgagtc
cccaccacag ggatcatcga ataccccttt gacttacaaa 660gtgtcatttt
cagaatggtc gatgtagggg gccaaaggtc agagagaaga aaatggatac
720actgctttga aaatgtcacc tctatcatgt ttctagtagc gcttagtgaa
tatgatcaag 780ttctcgtgga gtcagacaat gagaaccgaa tggaggaaag
caaggctctc tttagaacaa 840ttatcacata cccctggttc cagaactcct
cggttattct gttcttaaac aagaaagatc 900ttctagagga gaaaatcatg
tattcccatc tagtcgacta cttcccagaa tatgatggac 960cccagagaga
tgcccaggca gcccgagaat tcattctgaa gatgttcgtg gacctgaacc
1020cagacagtga caaaattatc tactcccact tcacgtgcgc cacagacacc
gagaatatcc 1080gctttgtctt tgctgccgtc aaggacacca tcctccagtt
gaacctgaag gagtacaatc 1140tggtctaatt gtgcctccta gacacccgcc
ctgcccttcc ctggtgggct attgaagata 1200cacaagaggg actgtatttc
tgtggaaaac aatttgcata atactaattt attgccgtcc 1260tggactctgt
gtgagcgtgt ccacagagtt tgtagtaaat attatgattt tatttaaact
1320attcagagga aaaacagagg atgctgaagt acagtcccag cacatttcct
ctctatcttt 1380tttttaggca aaaccttgtg actcagtgta ttttaaattc
tcagtcatgc actcacaaag 1440ataagacttg tttctttctg tctctctctc
tttttctttt ctatggagca aaacaaagct 1500gatttccctt ttttcttccc
ccgctaattc atacctccct cctgatgttt ttcccaggtt 1560acaatggcct
ttatcctagt tccattcttg gtcaagtttt tctctcaaat gatacagtca
1620ggacacatcg ttcgatttaa gccatcatca gcttaattta agtttgtagt
ttttgctgaa 1680ggattatatg tattaatact tacggtttta aatgtgttgc
tttggataca cacatagttt 1740cttttttaat agaatatact gtcttgtctc
actttggact gggacagtgg atgcccatct 1800aaaagttaag tgtcatttct
tttagatgtt taccttcagc catagcttga ttgctcagag 1860aaatatgcag
aaggcaggat caaagacaca caggagtcct ttcttttgaa atgccacgtg
1920ccattgtctt tcctcccttc tttgcttctt tttcttaccc tctctttcaa
ttgcagatgc 1980caaaaaagat gccaacagac actacattac cctaatggct
gctacccaga acctttttat 2040aggttgttct taattttttt gttgttgttg
ttcaagcttt tcctttcttt tttttcttgg 2100tgtttgggcc acgattttaa
aatgactttt attatgggta tgtgttgcca aagctggctt 2160tttgtcaaat
aaaatgaata cgaacttaaa aaataaaaaa aaaaaaaaaa aaaaa 221513359PRTHomo
sapiens 13Met Thr Leu Glu Ser Ile Met Ala Cys Cys Leu Ser Glu Glu
Ala Lys1 5 10 15Glu Ala Arg Arg Ile Asn Asp Glu Ile Glu Arg Gln Leu
Arg Arg Asp 20 25 30Lys Arg Asp Ala Arg Arg Glu Leu Lys Leu Leu Leu
Leu Gly Thr Gly 35 40 45Glu Ser Gly Lys Ser Thr Phe Ile Lys Gln Met
Arg Ile Ile His Gly 50 55 60Ser Gly Tyr Ser Asp Glu Asp Lys Arg Gly
Phe Thr Lys Leu Val Tyr65 70 75 80Gln Asn Ile Phe Thr Ala Met Gln
Ala Met Ile Arg Ala Met Asp Thr 85 90 95Leu Lys Ile Pro Tyr Lys Tyr
Glu His Asn Lys Ala His Ala Gln Leu 100 105 110Val Arg Glu Val Asp
Val Glu Lys Val Ser Ala Phe Glu Asn Pro Tyr 115 120 125Val Asp Ala
Ile Lys Ser Leu Trp Asn Asp Pro Gly Ile Gln Glu Cys 130 135 140Tyr
Asp Arg Arg Arg Glu Tyr Gln Leu Ser Asp Ser Thr Lys Tyr Tyr145 150
155 160Leu Asn Asp Leu Asp Arg Val Ala Asp Pro Ala Tyr Leu Pro Thr
Gln 165 170 175Gln Asp Val Leu Arg Val Arg Val Pro Thr Thr Gly Ile
Ile Glu Tyr 180 185 190Pro Phe Asp Leu Gln Ser Val Ile Phe Arg Met
Val Asp Val Gly Gly 195 200 205Gln Arg Ser Glu Arg Arg Lys Trp Ile
His Cys Phe Glu Asn Val Thr 210 215 220Ser Ile Met Phe Leu Val Ala
Leu Ser Glu Tyr Asp Gln Val Leu Val225 230 235 240Glu Ser Asp Asn
Glu Asn Arg Met Glu Glu Ser Lys Ala Leu Phe Arg 245 250 255Thr Ile
Ile Thr Tyr Pro Trp Phe Gln Asn Ser Ser Val Ile Leu Phe 260 265
270Leu Asn Lys Lys Asp Leu Leu Glu Glu Lys Ile Met Tyr Ser His Leu
275 280 285Val Asp Tyr Phe Pro Glu Tyr Asp Gly Pro Gln Arg Asp Ala
Gln Ala 290 295 300Ala Arg Glu Phe Ile Leu Lys Met Phe Val Asp Leu
Asn Pro Asp Ser305 310 315 320Asp Lys Ile Ile Tyr Ser His Phe Thr
Cys Ala Thr Asp Thr Glu Asn 325 330 335Ile Arg Phe Val Phe Ala Ala
Val Lys Asp Thr Ile Leu Gln Leu Asn 340 345 350Leu Lys Glu Tyr Asn
Leu Val 3551470DNAArtificial SequenceP1 Forward amplicon
w/barcodemisc_featuren=any nucleotidemisc_feature(34)..(44)n is a,
c, g, or t 14acactctttc cctacacgac gctcttccga tctnnnnnnn nnnngggtat
tcgatgatcc 60ctgtggtggg 701561DNAArtificial SequenceP2 Reverse
amplicon 15ctcggcattc ctgctgaacc gctcttccga tctcctttcc gtagacagct
ttggtgtgat 60g 611658DNAArtificial SequenceP3 Sequencing adapter 1
16aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct
581761DNAArtificial SequenceP4 Sequencing adapter 2 17caagcagaag
acggcatacg agatcggtct cggcattcct gctgaaccgc tcttccgatc 60t
611833DNAArtificial SequenceP1_1 5' fragment of SEQ ID NO14 (P1)
18acactctttc cctacacgac gctcttccga tct 331926DNAArtificial
SequenceP1_2 3' fragment of SEQ ID NO14 (P1) 19gggtattcga
tgatccctgt ggtggg 26
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References