(en)The present invention is directed to methods of diagnosing Noonan-like syndrome with loose anagen hair comprising detecting a mutation in SHOC2 gene. One specific diagnostic mutation disclosed is an A-to-G transition at position 4 resulting in a mutation at position 2 of SHOC2 amino acid sequence from serine to glycine. The invention also provides related sequences and kits.

1.ApplicationNumber: US-77539610-A
1.PublishNumber: US-8808992-B2
2.Date Publish: 20140819
3.Inventor: GELB BRUCE D.
TARTAGLIA MARCO
PENNACCHIO LEN
IYENGAR RAVI
MA'AYAN AVI

4.Inventor Harmonized: GELB BRUCE D(US)
TARTAGLIA MARCO(IT)
PENNACCHIO LEN(US)
IYENGAR RAVI(US)
MA AYAN AVI(US)

5.Country: US
6.Claims:
(en)The present invention is directed to methods of diagnosing Noonan-like syndrome with loose anagen hair comprising detecting a mutation in SHOC2 gene. One specific diagnostic mutation disclosed is an A-to-G transition at position 4 resulting in a mutation at position 2 of SHOC2 amino acid sequence from serine to glycine. The invention also provides related sequences and kits.

7.Description:
(en)This application claims priority from U.S. Provisional Application No. 61/215,568, filed May 6, 2009, which is hereby incorporated by reference in its entirety.


GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under grants 5R01HL71207, HD01294 and HL074728 awarded by the National Institutes of Health. The government has certain rights in the invention.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 2, 2010, is named 27527-0064001.txt, and is 18,828 bytes in size.
TECHNICAL FIELD OF THE INVENTION
The present invention is directed to methods of diagnosing Noonan-like syndrome with loose anagen hair comprising detecting a mutation in SHOC2 gene. One specific diagnostic mutation disclosed is an A-to-G transition at position 4 resulting in a mutation at position 2 of SHOC2 amino acid sequence from serine to glycine. The invention also provides related sequences and kits.
BACKGROUND OF THE INVENTION
Dysregulation of the RAS-MAPK signalling pathway has recently been recognized as the molecular cause underlying a group of clinically related developmental disorders with features including reduced postnatal growth, facial dysmorphism, cardiac defects, ectodermal anomalies, variable cognitive deficits and susceptibility to certain malignancies 8,9 . These Mendelian traits, including Noonan, LEOPARD, cardiofaciocutaneous and Costello syndromes, neurofibromatosis type 1 and related phenotypes, are caused by mutations in genes encoding RAS proteins (KRAS and HRAS), downstream transducers (RAF1, BRAF, MEK1 and MEK2), or pathway regulators (PTPN11, SOS1, NF1 and SPRED1). For Noonan syndrome (NS), the most common of these disorders (1:2500 live births), mutations are observed in several of these RAS-MAPK signalling pathway genes, constituting approximately 70% of cases.
Noonan syndrome (NS) is an autosomal dominant, pleiomorphic disorder characterized by short stature, facial dysmorphia, congenital heart defects (e.g., most commonly pulmonic stenosis and hypertrophic cardiomyopathy) and skeletal anomalies (Noonan, Am. J. Dis. Child. 116:373-80, 1968; Allanson, J. Med. Genet. 24:9-13, 1987). Other frequently associated disorders include a webbed neck, chest deformities, cryptorchidism, mental retardation, and bleeding diatheses. NS is a relatively common syndrome with an estimated incidence of 1:1000 to 1:2500 live births. The disorder is genetically heterogeneous and previously identified genes account for approximately only 65% of cases. In addition, there are some closely related disorders, such as Noonan-like syndrome with loose anagen hair, that are difficult to discern, particularly in infants and young children.
Noonan-like syndrome with loose anagen hair refers to disorders described under Online Mendelian Inheritance in Man database of John Hopkins University Accession No. OMIM 607721. Commonly, Noonan-like syndrome with loose anagen hair is characterized by one or more of the following phenotypic features: short stature, certain facial phenotype including high forehead, hypertelorism, palpebral ptosis and low-set and posteriorly rotated ears, macrocephaly, enlarged cerebral spinal fluid spaces, short neck with redundant skin, severe growth hormone (GH) deficiency, mild psychomotor delay with attention deficit/hyperactivity disorder (ADHD), mild dilatation of the pulmonary root, ectodermal abnormalities such as ichthyosis, darkly pigmented and hairless skin, and the unusual aspect of the hair, defined as loose anagen hair syndrome. Reviewed in Mazzanti et al., Am J Med Genet A. 2003, 118A:279-286.
The clinical diagnosis of NS and related disorders such as Noonan-like syndrome with loose anagen hair depends on recognition of the symptoms by a knowledgeable doctor. Nevertheless, substantial phenotypic variations, including mild or subtle cases, make the diagnosis difficult. Furthermore, the facial characteristics become less apparent with progressing age, so the condition will sometimes remain undiagnosed. No genetic test is currently available for diagnosing Noonan-like syndrome with loose anagen hair. Furthermore, currently available genetic tests for diagnosing NS (detecting mutations in PTPN11 and KRAS) account for only 50% of patients suspected of having NS. Therefore, there remains a great need for more specific (e.g., genetic) diagnostics of Noonan-like syndrome with loose anagen hair and other NS-related diseases.
SUMMARY OF THE INVENTION
As specified above, there remains a great need for more specific (e.g., genetic) diagnostics of Noonan-like syndrome with loose anagen hair and other NS-related diseases.
The present invention addresses these and other needs by providing a novel method for diagnosing Noonan-like syndrome with loose anagen hair in a human subject, comprising detecting a mutation in a SHOC2 nucleic acid molecule from the subject, wherein the mutation results in a SHOC2 protein comprising a glycine (Gly) substitution at position 2 of the SHOC2 amino acid sequence, and wherein the presence of said mutation in said SHOC2 nucleic acid molecule is diagnostic of Noonan-like syndrome with loose anagen hair in said human subject.
According to the method of the invention, such diagnostic mutation in a SHOC2 nucleic acid molecule can be detected by any method. In one embodiment, the mutation is detected by a method selected from the group consisting of hybridization (including solution and solid-phase hybridization methods), PCR amplification of a single specified genomic region, microarray-based sequencing, HPLC (including denaturing HPLC (DHPLC)), Denaturing Gradient Gel Electrophoresis (DGGE), Single Strand Conformation Polymorphism (SSCP), HOT cleavage, direct capture-based methods, next generation sequencing, exome sequencing, and whole genome sequencing.
In a preferred embodiment, the diagnostic mutation in a SHOC2 nucleic acid molecule associated with Noonan-like syndrome with loose anagen hair is an A to G transition at position 4 of the SHOC2 coding sequence. In one embodiment, the sequence of the wild-type human SHOC2 nucleic acid is SEQ ID NO: 1, which corresponds to GenBank Accession No. NM — 007373, wherein the coding sequence for SHOC2 protein begins at position 350, and the mutant human SHOC2 nucleic acid sequence associated with Noonan-like syndrome with loose anagen hair (SEQ ID NO: 2) has the G substitution at position 4 of the coding sequence of NM — 007373 (i.e., position 353). In one embodiment, the wild-type human SHOC2 protein sequence is SEQ ID NO: 3, which corresponds to GenBank Accession No. NP — 031399, and the mutant human SHOC2 protein sequence associated with Noonan-like syndrome with loose anagen hair (SEQ ID NO: 4) has the serine (Ser)→glycine (Gly) (Ser2Gly or S2G) substitution at position 2 of NP — 031399.
The present invention is also directed to kits for diagnosing Noonan-like syndrome with loose anagen hair, comprising one or more oligonucleotides that specifically hybridize to (or hybridize adjacent to) a site of mutation of a SHOC2 nucleic acid molecule, wherein the mutation results in an amino acid substitution in a SHOC2 polypeptide encoded by the SHOC2 nucleic acid molecule; and instructions for use, wherein the amino acid substitution in the SHOC2 polypeptide is at position 2 of the SHOC2 amino acid sequence. In one embodiment, the amino acid substitution is glycine for serine. In a further embodiment, the site of mutation comprises nucleotide 4 of the SHOC2 coding sequence. In a further embodiment, the mutation at nucleotide 4 of the SHOC2 coding sequence is an A to G transition.
In one specific embodiment, the kit of the invention comprises two oligonucleotides 5′-GTGTAGGATCTTTGTCTCTTC-3′ (SEQ ID NO: 5) and 5′-CCTTCTTTCCATCTTTGGCAT-3′ (SEQ ID NO: 6).
The present invention is also directed to an isolated SHOC2 polypeptide variant comprising a serine (Ser)→glycine (Gly) (Ser2Gly or S2G) substitution at position 2 of the SHOC2 amino acid sequence. In one embodiment, such isolated SHOC2 polypeptide variant has SEQ ID NO: 4. The instant invention is also directed to isolated nucleic acid molecules encoding such a SHOC2 variant. In one embodiment, such nucleic acid molecule has SEQ ID NO: 2.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 . The germline 4A>G mutation in the SHOC2 gene underlies a distinctive phenotype of the neuro-cardio-facial-cutaneous syndrome family. (a) DHPLC profiles showing the de novo origin of the 4A>G nucleotide change in two affected subjects (left), and electropherograms documenting the heterozygous condition for this mutation at codon 2 in peripheral leukocytes, skin fibroblasts, buccal epithelial cells and/or hair bulb cells from these individuals. (b) Representative phenotypic features of affected subjects carrying the SHOC2 mutation. (c) SHOC2 genomic organization and protein structure. The coding exons are shown at the top as numbered filled boxes. Intronic regions are reported as dotted lines. SHOC2 motifs comprise an N-terminal lysine-rich region (grey coloured; Prosite motif score=8.8, http://www.expasy.ch/tools/scanprosite/) followed by 19 leucine-rich repeats (Pfam hits with an E-value<0.5 are black coloured, while those with an E-value>1 are represented in white, http://pfam.janelia.org/). Numbers above the domain structure indicate the amino acid boundaries of those domains.
FIG. 2 . The disease-causing 4A>G change in SHOC2 promotes protein myristoylation and cell membrane targeting. (a) [ 3 H]myristic acid incorporation (middle) occurs in SHOC2 S2G but not in SHOC2 wt or SHOC2 S2A . Equivalent levels of SHOC2 proteins in immunoprecipitates (left) and [ 3 H]myristic acid incorporation in cells (right) are shown. (b) SHOC2 wt is uniformly spread in the cytoplasm and nucleus in starved Cos-1 cells (upper left) and is restricted to the nucleus following EGF stimulation (upper right), while SHOC2 S2G is targeted to the cell membrane basally (lower left) and after stimulation (lower right). Confocal laser microscopy visualized SHOC2 (anti-V5 monoclonal antibody, then Alexa Fluor-594 goat anti-mouse antibody; red), actin cytoskeleton (Alexa Fluor 488-phalloidin; green) and nuclei (DAPI; blue). Images are single optical sections representative of >50 transfected cells for each experiment. Bars indicate 20 μm. (c) Cell fractioning assay documenting preferential membrane targeting of SHOC2 S2G . Transiently transfected cells were serum-starved or stimulated with EGF, and lysates were fractionated to separate membrane-associated proteins. ERBB2 is shown to demonstrate equivalent fractionation efficiency, while anti-V5 blot from cell lysates show equivalent transfection efficiency.
FIG. 3 . Functional characterization of the disease-causing 4A>G change in SHOC2. (a) Subcellular localization of co-expressed SHOC2 wt (green) and SHOC2 S2G (red) documenting that SHOC2 S2G does not impair EGF-stimulated SHOC2 wt translocation to the nucleus. Imaging of V5-tagged (anti-V5 monoclonal antibody, then Alexa Fluor-594 goat anti-mouse antibody) and Myc-tagged (anti-Myc antibody, then Alex Fluor 488 goat anti-rabbit antibody) SHOC2 proteins and nuclei (DAPI, blue). Panels above show Myc-tagged SHOC2 wt and V5-tagged SHOC2 S2G and below show V5-tagged SHOC2 wt and Myc-tagged SHOC2 S2G . Cells were imaged basally (left) and following EGF stimulation (right). Bars indicate 20 μm. (b) Lysates of Cos-1 cells co-expressing Myc-tagged SHOC2 wt and V5-tagged SHOC2 S2G were immunoprecipitated using anti-Myc (above panel) or anti-V5 (below panel) antibody, and immunoprecipitated proteins were visualized by western blotting. These results indicate that SHOC2 proteins do not form heterodimers. (c) ERK phosphorylation in V5-tagged SHOC2 wt or SHOC2 S2G transiently expressed Neuro2A cells basally or following EGF stimulation.
FIG. 4 . Consequences of SHOC2 S2G expression in C. elegans vulva development. Nomarski images of vulvas of adult animals (a-d). A normal vulva is observed in animals expressing SHOC2 wt (a), while in worms expressing SHOC2 S2G (b and c) or myr::SHOC2 wt (d) a protrusion of the vulva is visible. Subcellular localization of V5-tagged SHOC2 proteins in excretory canal cells (e-g) and intestinal cells (h-j). In both cell types, SHOC2 wt protein is present throughout the cytoplasm (e and h), while both SHOC2 S2G (f and i) and myr::SHOC2 wt (g and j) are enriched in or restricted to the plasma membrane. Confocal analysis was performed by immunofluorescence staining using an Anti-V5 antibody (red). In intestinal cells, nuclei express GFP due to pelt-2::GFP plasmid used as a marker for transformation. Nomarski images of VPCs at L3 stage (k-n). In animals expressing SHOC2 wt only P6.p descendant invaginate (k), while in SHOC2 S2G (l and m) and myr::SHOC2 wt (n) expressing animals also P5.p (l to n) and P7.p descendants (m and n) detach from the cuticle. Anterior is to the left and dorsal is up in all images.
FIG. 5 . Mammalian protein-protein interaction network analysis identifies SHOC2 as the best disease gene candidate for Noonan syndrome or a clinically related phenotype. The network was constructed by connecting proteins known to be mutated in Noonan syndrome (cyan) through two nodes/three links using the human interactome. SHOC2, the best candidate, is shown in magenta. Connections indicate protein-protein interactions. Leading candidates and Z scores are reported in Supplementary Table 1.
FIG. 6 . Subcellular localization of the Myc-tagged SHOC2 wt and SHOC2 S2G proteins expressed in Cos-1 cells, basally and following EGF stimulation. SHOC2 wt is uniformly spread in the cytoplasm and nucleus in starved cells (upper left), and is restricted to the nucleus following stimulation (upper right), while the SHOC2 S2G is targeted to the cell membrane in both basal (lower left) and stimulated (lower right) conditions. Confocal laser scanning microscopy was performed using anti-Myc monoclonal antibody, followed by Alexa Fluor-594 goat anti-mouse antibody (red), while actin cytoskeleton was detected by Alexa Fluor 488-phalloidin (green). Nuclei are visualized by DAPI staining (blue). Co-localization areas were detected in yellow. Images represent single optical sections representative of >50 transfected cells observed in each experiment. Bars indicate 20 μm.
FIG. 7 . Subcellular localization of the V5-tagged SHOC2 wt and SHOC2 S2G proteins expressed in Neuro2A cells, basally and following EGF stimulation. SHOC2 wt is uniformly spread in the cytoplasm and nucleus in starved cells (upper left), and is restricted to the nucleus following stimulation (upper right), while the SHOC2 S2G is targeted to the cell membrane in both basal (lower left) and stimulated (lower right) conditions. Confocal laser scanning microscopy was performed using anti-V5 monoclonal antibody, followed by Alexa Fluor-594 goat anti-mouse antibody (red), while actin cytoskeleton was detected by Alexa Fluor 488-phalloidin (green). Nuclei are visualized by DAPI staining (blue). Co-localization areas were detected in yellow. Images represent single optical sections representative of >50 transfected cells observed in each experiment. Bars indicate 8 μm.
FIG. 8 . Myristoylation is required for membrane targeting of the SHOC2 S2G mutant. V5-tagged SHOC2 S2G is membrane-targeted in untreated Cos-1 cells (top), while membrane targeting is progressively lost in cells treated with an NMT inhibitor (200 μM, middle; 300 μM, bottom). Confocal laser scanning microscopy was performed using anti-V5 monoclonal antibody, followed by Alexa Fluor-594 goat anti-mouse antibody (red), while actin cytoskeleton was detected by Alexa Fluor 488-phalloidin (green). Nuclei are visualized by DAPI staining (blue). Co-localization areas were detected in yellow. Images represent single optical sections representative of >50 transfected cells observed in each experiment. Bars indicate 20 μm.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on an unexpected discovery that aberrantly acquired N-myristoylation of SHOC2, a cytoplasmic leucine-rich repeat-containing protein that positively modulates RAS-mitogen activated protein kinase (MAPK) signal flow, 3-6 underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. As demonstrated in the Examples section, below, twenty-five subjects with a relatively consistent phenotype termed Noonan-like syndrome with loose anagen hair [OMIM (Online Mendelian Inheritance in Man database of John Hopkins University) 607721] 7 shared an A-to-G transition at position 4 in SHOC2 coding sequence that leads to serine (Ser)→glycine (Gly) substitution at position 2 of SHOC2 protein (Ser2Gly or S2G) and introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 protein to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of mutant SHOC2 protein in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of mutant SHOC2 in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signalling. These results provide the first example of an acquired co-translational modification leading to gain of function and causing human disease.
In one embodiment, the sequence of the wild-type human SHOC2 nucleic acid is SEQ ID NO: 1, which corresponds to GenBank Accession No. NM — 007373, wherein the coding sequence for SHOC2 protein begins at position 350, and the mutant human SHOC2 nucleic acid sequence associated with Noonan-like syndrome with loose anagen hair (SEQ ID NO: 2) has the G substitution at position 4 of the coding sequence of NM — 007373 (i.e., position 353). In one embodiment, the wild-type human SHOC2 protein sequence is SEQ ID NO: 3, which corresponds to GenBank Accession No. NP — 031399, and the mutant human SHOC2 protein sequence associated with Noonan-like syndrome with loose anagen hair (SEQ ID NO: 4) has the serine (Ser)→glycine (Gly) (Ser2Gly or S2G) substitution at position 2 of NP — 031399.
The subject to whom the diagnostic applications of this disclosure are directed may be any mammal. In a preferred embodiment, the subject is a human. The subject may be of any age (e.g., an adult, a child, an infant), which includes prenatal diagnostics.
In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
DEFINITIONS
As used herein, the term “Noonan-like syndrome with loose anagen hair” refers to disorders described under Online Mendelian Inheritance in Man database of John Hopkins University Accession No. OMIM 607721. Commonly, Noonan-like syndrome with loose anagen hair is characterized by one or more of the following phenotypic features: short stature, certain facial phenotype including high forehead, hypertelorism, palpebral ptosis and low-set and posteriorly rotated ears, macrocephaly, enlarged cerebral spinal fluid spaces, short neck with redundant skin, severe growth hormone (GH) deficiency, mild psychomotor delay with attention deficit/hyperactivity disorder (ADHD), mild dilatation of the pulmonary root, a unique combination of ectodermal abnormalities including ichthyosis, darkly pigmented and hairless skin, and the unusual aspect of the hair, defined as loose anagen hair syndrome. Reviewed in Mazzanti et al., Am J Med Genet A. 2003, 118A:279-286.
As used herein, the term “Noonan syndrome” or “NS” refers to disorders and diseases described under Accession No. OMIM 163950.
“N-myristoylation” is a common form of protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. 2
The term “SHOC2 nucleic acid molecule” refers to a nucleic acid molecule comprising a nucleotide sequence encoding a SHOC2 protein. The terms “SHOC2 coding nucleic acid sequence” or “SHOC2 coding sequence” refer to a portion of a SHOC2 nucleic acid molecule which encodes a SHOC2 protein.
The terms “mutant” and “mutation” mean any detectable change in genetic material or any product, process, mechanism, or result of such a change. When compared to a control material, such change may be also referred to as a “variant” or an “abnormality”. This includes gene mutations, in which the structure of a gene is altered, arising from any mutation process, and the structure and/or amount of any expression product (e.g., RNA and/or protein) expressed by such a modified gene is also altered.
As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of at least 10 nucleotides, preferably of at least 15 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or biotin. In one embodiment, an oligonucleotide can be used as a probe to detect the presence of a mutant nucleic acid. A library of oligonucleotides arranged on a solid support, such as a silicon wafer or chip, can be used to detect various mutations of interest. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
Diagnostic Methods of the Invention
The present invention is directed to methods for diagnosing Noonan-like syndrome with loose anagen hair in a human subject, comprising detecting a mutation in a SHOC2 nucleic acid molecule from the subject, wherein the mutation results in an SHOC2 polypeptide comprising a glycine substitution at position 2 of the SHOC2 amino acid sequence, and wherein the presence of said mutation in said SHOC2 nucleic acid molecule is diagnostic of Noonan-like syndrome with loose anagen hair in said human subject. In a preferred embodiment, the mutation in the SHOC2 nucleic acid molecule is an A to G transition at position 4 of the SHOC2 nucleic acid sequence.
The above mutation can be detected by any acceptable method. Cost-efficient fast methods suitable for high-throughput use are particularly preferred. Non-limiting examples of such methods include, e.g., solution or solid-phase hybridization methods, PCR amplification of a single specified genomic region, microarray-based sequencing, HPLC (e.g., denaturing HPLC (DHPLC)), Denaturing Gradient Gel Electrophoresis (DGGE), Single Strand Conformation Polymorphism (SSCP), HOT cleavage, direct capture-based methods, next generation sequencing, exome sequencing, and whole genome sequencing.
Denaturing HPLC (DHPLC) can separate heteroduplexes that differ by as little as one base pair. Thus, the use of DHPLC can be applied to point mutation detection (Underhill et al., Genome Research 7:996, 1997; Liu et al., Nucleic Acid Res. 26; 1396, 1998). DHPLC analyses are carried out at a partially denaturing temperature, i.e., a temperature sufficient to denature a heteroduplex at the site of base pair mismatch, homoduplexes can be separated from heteroduplexes having the same base pair length (Hayward-Lester et al., Genome Research 5:494, 1995; Underhill et al., Proc. Nat'l. Acad. Sci. USA 93:193, 1996; Doris et al., DHPLC Workshop, 1997, Stanford University). “Matched Ion Polynucleotide Chromatography” (MIPC), or Denaturing “Matched Ion Polynucleotide Chromatography” (DMIPC) as described in U.S. Pat. No. 6,287,822 or 6,024,878, are separation methods that can also be useful in connection with the present disclosure.
Denaturing Gradient Gel Electrophoresis (DGGE) is a method for resolving two DNA fragments of identical length on the basis of sequence differences as small as a single base pair change, using electrophoresis through a gel containing varying concentrations of denaturant (Guldberg et al., Nuc. Acids Res. 1994, 22:880).
Single Strand Conformation Polymorphism (SSCP) is a method for detecting sequence differences between two DNAs, comprising hybridization of the two species with subsequent mismatch detection by gel electrophoresis (Ravnik-Glavac et al., Hum. Mol. Genet. 3:801, 1994).
“HOT cleavage” is a method for detecting sequence differences between two DNAs, comprising hybridization of the two species with subsequent mismatch detection by chemical cleavage (Cotton, et al., Proc. Natl. Acad. Sci. USA 85:4397, 1988).
The above methods are preferably followed by direct sequencing.
More recently developed techniques using microarrays, preferably microarray techniques allowing for high-throughput screening, can also be advantageously implemented for detecting mutations. Microarrays may be designed so that the same set of identical oligonucleotides is attached to at least two selected discrete regions of the array, so that one can easily compare a normal sample, contacted with one of the selected regions of the array, against a test sample, contacted with another of the selected regions. These arrays avoid the mixture of normal sample and test sample, using microfluidic conduits. Useful microarray techniques include those developed by Nanogen, Inc (San Diego, Calif.) and those developed by Affymetrix. However, all types of microarrays, also called “gene chips” or “DNA chips”, may be adapted for the identification of mutations. Such microarrays are well known in the art (see, for example, the following: U.S. Pat. Nos. 6,045,996; 6,040,138; 6,027,880; 6,020,135; 5,968,740; 5,959,098; 5,945,334; 5,885,837; 5,874,219; 5,861,242; 5,843,655; 5,837,832; 5,677,195 and 5,593,839). In principal, any type of array, for example, dot blots on a nylon hybridization membrane (see Sambrook et al., Molecular Cloning A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) could be used, although, as will be recognized by those of skill in the art, very small arrays will be preferred because hybridization volumes will be smaller. For these assays nucleic acid hybridization and wash conditions are chosen so that the attached oligonucleotides “specifically bind” or “specifically hybridize” to at least a portion of a SHOC2 nucleic acid molecule present in a target sample, i.e., the probe hybridizes, duplexes or binds to the SHOC2 locus with a complementary nucleic acid sequence but does not hybridize to a site with a non-complementary nucleic acid sequence. As used herein, one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules. It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see, e.g., Shalon et al., supra, and Chee et al., Science 274:610-4, 1996).
A variety of methods are available for detection and analysis of a hybridization event. Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used to label a probe (e.g., oligonucleotide), detection and analysis are carried out fluorimetrically, calorimetrically or by autoradiography. By observing and measuring emitted radiation, such as fluorescent radiation or a particle emission, information may be obtained about hybridization events. When fluorescently labeled probes are used, the fluorescence emissions at each site of array can, preferably be detected by scanning confocal laser microscopy. In one embodiment, a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used. Alternatively, a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al., Genome Res. 6:639-695, 1996).
For description of direct capture-based methods, next generation sequencing, exome sequencing (targeted sequencing of all protein-coding regions), and whole genome sequencing see, e.g., Olson, Nat Methods 2007, 4:891-892; Turner et al., Nat Methods 2009, 6:315-316; Okou et al., Nat Methods 2007, 4:907-909; Albert et al., Nat Methods 2007, 4:903-905; Hodges et al., Nat Genet. 2007, 39:1522-1527; Ng et al., Nat. Genet., 2010, 42:30; Ng et al., Nature, 2009, 461:272; Choi et al., PNAS, 2009, 106:19096; Drmanac et al., Science, 2010, 327:78
The present invention is also directed to kits for diagnosing Noonan-like syndrome with loose anagen hair, comprising an oligonucleotide that specifically hybridizes to (or hybridizes adjacent to) a site of mutation of a SHOC2 nucleic acid molecule, wherein the mutation results in an amino acid substitution in a SHOC2 polypeptide encoded by the SHOC2 nucleic acid molecule; and instructions for use, wherein the amino acid substitution in the SHOC2 polypeptide is at position 2 of the SHOC2 amino acid sequence. In one embodiment, the amino acid substitution is glycine for serine. In a further embodiment, the site of mutation comprises nucleotide 4 of the SHOC2 nucleic acid molecule. In a further embodiment, the mutation at site 4 is an A to G transition.
In one specific embodiment, the kit of the invention comprises two oligonucleotides 5′-GTGTAGGATCTTTGTCTCTTC-3′ (SEQ ID NO: 5) and 5′-CCTTCTTTCCATCTTTGGCAT-3′ (SEQ ID NO: 6).
Example 1
To rationalize further candidate gene approaches to NS gene discovery, a systems biology approach was used based on in silico protein network analysis. By applying a graph theory algorithm on a filtered consolidated human interactome, a subnetwork of proteins generated from an integrated network of mammalian protein interaction databases and cell-signalling network datasets was derived by seeding with the known RAS/MAPK mutant proteins ( FIG. 5 ).
To identify potential NS disease genes, Z scores were computed using a binomial proportions test, ranking the significance of the intermediate nodes based on their connections to the seed proteins 10 (Table 1).





TABLE 1



Top Noonan syndrome disease candidates predicted by mammalian

protein-protein interaction network analysis.








Protein
Links for
Total links in
Links
Total links in


name
the node
background
to seed
subnetwork
z-score








One node/Two links subnetwork











SHOC2
4
11184
2
22
22.48034

NRAS
25
11184
2
22
8.80567

CRKL
34
11184
2
22
7.48228

JAK1
39
11184
2
22
6.95066

INSR
41
11184
2
22
6.76514

JAK2
60
11184
2
22
5.48344

BCL2
68
11184
2
22
5.10772

PRKCA
91
11184
2
22
4.30827

EGFR
106
11184
2
22
3.92713






Two nodes/Three links subnetwork











SHOC2
4
11184
2
223
6.86825

RASSF2
2
11184
1
223
4.85658

SPRY1
3
11184
1
223
3.88303

FRS2
11
11184
2
223
3.84067

SLAMF6
4
11184
1
223
3.29149

APBB1IP
4
11184
1
223
3.29149

GRAP
5
11184
1
223
2.88021

HRAS
5
11184
1
223
2.88021

RIT2
7
11184
1
223
2.32640

NRAS
25
11184
2
223
2.14823

RRAS
8
11184
1
223
2.12572
























































The top candidates were curated to exclude genes previously screened or those with inappropriate expression patterns. Resequencing of coding exons for the best candidate, SHOC2, in a NS cohort including 96 individuals who were negative for mutations in previously identified disease genes and opportunely selected to represent the wide phenotypic spectrum characterizing this disorder revealed an A-to-G transition at position 4 of the gene, predicting the Ser2Gly amino acid substitution (S2G), in four unrelated individuals ( FIG. 1 a ). This nucleotide change had not been reported in a public SNP database, and Ser 2 in SHOC2 is conserved among vertebrate orthologues. All cases were sporadic, and genotyping of parental DNAs available for three of the four subjects documented the absence of the sequence variant in the parents and confirmed paternity in each family, providing evidence that the change was a de novo mutation associated with the disease. For these subjects, DNAs from several tissues were available and all harboured the S2G mutation, providing evidence that the defect was inherited through the germline ( FIG. 1 a ). SHOC2 in a cohort of 410 mutation-negative subjects with NS or a related phenotype was then analyzed. 21 with the 4A>G missense change were observed and proved that mutations were de novo in twelve families from which parental DNAs were available. No additional disease-associated SHOC2 sequence variant was identified in the analyzed cohort, strongly suggesting a specific pathogenetic role for the S2G amino acid substitution.
Review of the features of the SHOC2 mutation-positive individuals revealed a relatively consistent phenotype, previously termed Noonan-like syndrome with loose anagen hair 7 ( FIG. 1 b ). Their features were at first view reminiscent of Noonan syndrome. Phenotype analysis of these subjects, however, was notable for the observation that they exhibited reduced growth associated with proven growth hormone (GH) deficiency, cognitive deficits, distinctive hyperactive behaviour, and hair anomalies including easily pluckable, sparse, thin, slow growing hair. In six subjects, a diagnosis of loose anagen hair (LAH) was confirmed by microscopic examination of pulled hairs, many of which were in the anagen phase but lacked an inner and outer root sheath. Most of them also exhibited hairless and darkly pigmented skin with eczema or ichthyosis. Voice was characteristically hypernasal. Cardiac anomalies were observed in the majority of the cases, with dysplasia of the mitral valve and septal defects significantly overrepresented compared with the general NS population. Overall, these subjects appeared to share a consistent phenotype that was characterized by an unusual combination of features observed in disorders of the neuro-cardio-facial-cutaneous disorders family. Mazzanti et al. 7 reported the new syndrome Noonan-like syndrome with loose anagen hair by detailing three children with short stature, the same facial phenotype, macrocephaly, enlarged spinal fluid space, short neck with redundant skin, severe GH deficiency, mild psychomotor delay with attention deficit/hyperactivity disorder, mild dilation of the pulmonary root in two of them and a unique combination of ectodermal abnormalities.
SHOC2 is a widely expressed protein composed almost entirely by leucine-rich repeats (LRR) and has a lysine-rich sequence at the N-terminus ( FIG. 1 c ). In C. elegans , where Shoc2/sur8 was discovered, the protein acts as a positive modulator of the signal transduction elicited by eg1-15 and let-23, and mediated by let-60, homologues of vertebrate FGFR, EGFR and RAS family members, respectively 3,4 . Since LRRs can provide a structural framework for protein-protein interactions, SHOC2 is believed to function as a scaffold linking RAS to downstream signal transducers 4-6 . Based on the N-terminal position of the S2G substitution, the present inventors hypothesized that co-translational processing might be perturbed in the mutant protein, making it a substrate for the N-myristoyltransferase (NMT). N-terminal myristoylation is an irreversible modification occurring co-translationally in which myristate, a 14-carbon saturated fatty acid, is covalently added to an N-terminal glycine residues after excision of the initiator methionine residue by methionylaminopeptidase 1,2 . Glycine at codon 2 is absolutely required, small uncharged residues at positions 3 and 6 are generally needed, and basic residues at positions 8 and 9 are preferred 11 . Save the presence of Ser at position 2, the N-terminal sequence of the SHOC2 satisfied those consensus rules, and in silico analysis predicted myristoylation of the SHOC2 S2G mutant with high confidence. To verify this, the myristoylation status of wild type and mutant SHOC2 proteins transiently expressed in Cos-1 cells was evaluated ( FIG. 2 a ). SHOC2 S2G incorporated [ 3 H]-myristic acid, while the wild type protein and the disease-unrelated SHOC2 S2A did not.
N-myristoylation facilitates anchoring of proteins to intracellular membranes. To explore whether it conferred membrane targeting to mutant SHOC2, the subcellular localization of tagged SHOC2 proteins was analyzed in Cos-1 cells ( FIG. 2 b and FIG. 6 ). Confocal laser microscopy analysis documented that SHOC2 wt was uniformly distributed in the cytoplasm and nucleus in starved cells but was restricted to the nucleus following EGF stimulation, implying an unexpected role for this protein in signal transduction. In contrast, SHOC2 S2G was specifically targeted to the cell membrane in both states. This aberrant localization of SHOC2 S2G was confirmed using 293T and Neuro2A cell lines ( FIG. 7 ) and by cell fractionation ( FIG. 2 c ). Treatment with 2-hydroxymyristic acid, an NMT inhibitor, at varying doses reduced or abolished SHOC2 S2G 's membrane localization ( FIG. 8 ), confirming a dependency upon myristoylation. In addition, even in the absence of efficient myristoylation, the mutant did not translocate to the nucleus upon EGF stimulation, indicating possible loss of function. To exclude the possibility that SHOC2 S2G might play a dominant negative effect by sequestering the wild-type protein to the cell membrane, impairing its EGF-dependent translocation to the nucleus, heterodimerization of the wild type and SHOC2 S2G proteins was assayed by confocal microscopy and co-immunoprecipitation assays in COS-1 cells transiently co-transfected with V5- and Myc-tagged proteins ( FIG. 3 a,b ). These experiments demonstrated that the wild-type and mutant proteins do not heterodimerize, ruling out that possibility. Next, it was explored whether SHOC2 S2G altered intracellular signalling through MAPK. Wild-type and mutant SHOC2 were expressed in Cos-1, 293T and Neuro2A cells. While no significant change in ERK activation in Cos-1 and 293T cells was observed, SHOC2 S2G expression promoted enhanced EGF-dependent ERK phosphorylation compared to wild type SHOC2, in neuroblastoma Neuro2A cells ( FIG. 3 c ).
To explore further the functional effects of the SHOC2 S2G mutant, C. elegans was used as an experimental model. In C. elegans , reduced Shoc2/Sur-8 (Sur8 rf ) causes no phenotype but can suppress the gain-of-function Ras (let-60 gof )-induced multivulva phenotype (Muv) 4 . It was tested whether expression of SHOC2 proteins could rescue the suppressed Muv phenotype in the sur-8 rf ; let-60 gof genetic background. While wild type SHOC2 was able to replace Sur-8 functionally, SHOC2 S2G failed to do so (STable 2). Expression of the mutant in let-60 gof worms did not suppress the Muv phenotype (Table 2), excluding dominant negative effects for SHOC2 S2G . In a wild-type genetic background, expression of SHOC2 S2G at embryonic and early larval stages of development caused no visible phenotype.





TABLE 2



Vulval precursor cell induction in C. elegans strains

expressing the wild-type or mutant SHOC2 protein.






Induction of vulval fate



VPC














Genotype
Transgene
N
Muv a
N
P3.p
P4.p
P5.p
P6.p
P7.p
P8.p
Average
















let-60 gof
none
135
68.1
39
30.8
41.0
100
100
100
30.8
4.0

let-60 gof ;
none
302
7.9
85
2.4
5.9
100
100
100
14.1
3.2

sur-8 rf

let-60 gof ;
SHOC2 wt
271
19.6 b
45
4.4
35.6 c
100
100
100
37.8
3.8 b

sur-8 rf

let-60 gof ;
SHOC2 S2G
104
10.6
29
3.4
3.4
100
100
100
3.4
3.1 d

sur-8 rf

wild type
none
>100
0

let-60 gof
none
135
68.1

let-60 gof
SHOC2 wt
72
61.1

let-60 gof
SHOC2 S2G
132
65.9



Strains: let-60 gof indicates let-60(n1046), sur-8 rf indicates sur-8(ku167).

SHOC2 wt indicates hsp16.2::SHOC2 wt ::V5, SHOC2 S2G indicates hsp16.2::SHOC2 S2G ::V5.

N indicates the number of animals scored, VPC indicates vulval precursor cell.

Animals were heat-shocked at early L3 stage. Induction of vulval fate is expressed as the percent of individual VPCs (P3.p to P8.p) dividing more than one time. Average refers to the average number of VPCs induced per animal.

P values were calculated using t-Student test for average VPC induction and z statistics for all other proportions.

a Muv is expressed as the percent of animals with ectopic pseudovulvae.

b Significantly different from the let-60 gof ; sur-8 rf strain (P < 0.001).

c Significantly different from the let-60 gof ; sur-8 rf strain (P < 0.005).

d Significantly different from the let-60 gof ; sur-8 rf strain expressing the SHOC2 wt transgene (P < 0.001).















































In contrast, its expression at early L3 stage caused abnormal vulval development, resulting in protruding vulva (Pv1), decreased egg laying efficiency (Eg1) and accumulation of larvae inside the mother with the formation of bag-of-worms adults (Bag phenotype) (Table 3 and FIG. 4 a - c ). These neomorphic phenotypes were absent in animals expressing SHOC2 wt but were also observed when SHOC2 wt tagged with an N-myristoylation sequence (myr::SHOC2 wt ) was expressed (Table 3 and FIG. 4 d ).





TABLE 3



Phenotypes resulting from expression of the

SHOC2 wt , SHOC2 S2G or myr::SHOC wt transgene.










Genotype
Transgene
N
Pvl
N
Egl
N
Bag












Wild type
none
56
1.8
25
19.2
20
5.0

Wild type
SHOC2 wt
44
0
25
16.2
20
5.0

Wild type
SHOC2 S2G
96
17.7 a
25
26.3 a
20
55.0 d

Wild type
myr::SHOC wt
49
36.7 b
25
25.5 c
20
45.0 e



Animals were heat-shocked at early L3 stage. SHOC2 wt indicates hsp16.2::SHOC2 wt ::V5, SHOC2 S2G indicates hsp16.2::SHOC2 S2G ::V5, myr::SHOC2 wt indicates hsp16.2::myr::SHOC2 wt ::V5.

N indicates the number of animals scored. Pvl is the percent of animals with a protruding vulva.

Egl is the average number of eggs per worm contained in the uterus. Bag is the percent of a bag-of-worms animals 6 days post fertilization.

a-e Significantly different from SHOC2 wt ( a P < 0.01; b P < 0.0001; c P < 0.05; d P < 0.005; e P < 0.02).






























The SHOC2 S2G and myr::SHOC2 wt proteins were targeted to the cell membrane in various C. elegans cell types, while SHOC2 wt was observed diffusely throughout the cytoplasm and nucleus ( FIG. 4 e - j ). The defects in vulva formation were not due to increased induction of the vulva cell fate in vulval precursor cells (VPC) as expression of SHOC2 S2G did not reduce the vulvaless phenotype of a let-23 rf hypomorph mutant (Table 4), nor increase the penetrance of the Muv phenotype of let-60 gf animals (Table 2). At the late L3/early L4 stage, vulva morphogenesis normally begins with the descendants of vulval precursor cell (VPC) P6.p detaching from the cuticle and forming a symmetric invagination. Animals in which the expression of SHOC2 wt had been induced at early L3 maintained this pattern. In contrast, in larvae expressing SHOC2 S2G (17/48) or myr::SHOC2 wt (10/22), descendants of VPCs P5.p and/or P7.p also detached from the cuticle, resulting in larger and asymmetric invaginations ( FIG. 4 k - n ). This morphogenesis defect was the earliest detectable neomorphic effect of the SHOC2 S2G mutation on vulval development and likely underlies the abnormalities seen in the adult vulva.





TABLE 4



Phenotypes observed in C. elegans let-23 rf mutants after

expression of the wild-type or mutant SHOC2 transgene.








Genotype
Transgene
N
Bag











wild type
none
24
0


let-23 rf
none
93
84.9


let-23 rf
SHOC2 wt
92
85.9


let-23 rf
SHOC2 S2G
93
95.7 a





let-23 rf indicates let-23(sy1), SHOC2 wt indicates hsp16.2::SHOC2 wt ::V5, SHOC2 S2G indicates hsp16.2::SHOC2 S2G ::V5.


Worms were heat-shocked at early L3 stage.


N indicates the number of animals scored. Bag is the percent of animals that become a “bag-of-worm” 4 days post-fertilization.


a Significantly different from the let-23 rf strain and the let-23 rf strain expressing SHOC2 wt (P < 0.05 in both comparisons).






























It is discovered herein that a SHOC2 mutation promoting N-myristoylation of its protein product causes Noonan-like syndrome with loose anagen hair. Acquired co-translational processing, a unique finding for inherited human disease, results in constitutive membrane targeting, leading to increased MAPK activation in a cell context-specific manner. Cell-specific RAS pathway activation has also been observed with NS-associated SHP-2 mutants. 12-14 While not well understood, this phenomenon explains why, despite the ubiquitousness of RAS signalling, development is perturbed in certain tissues in these disorders.
In C. elegans , N-myristoylated SHOC2 expression altered morphogenesis during vulval development, a process for which the involvement of Ras signalling is well established. Specification of VPCs was not altered, contrasting with what is noted with many other Ras pathway mutants. Rather, perturbation of the morphogenetic movements of the VPC descendant cells was observed. While numerous mutants altering vulval specification and morphogenesis have been identified, far less is known about processes affecting only morphogenesis. 15,16 It is possible that SHOC2 S2G alters Ras signalling in steps downstream of the induction of the vulval fate. Alternatively, SHOC2 S2G -induced vulva defects might arise through perturbation of signalling pathways other than Ras-MAPK, such as signalling mediated by the Rho GTPase, Rac, which is critical for vulval morphogenesis 17 .
A unique feature of the SHOC2 mutation is its association with loose anagen hair. This phenotype occurs in isolation or with NS and has been without molecular cause. Hair shafts from affected individuals show features of the anagen stage of hair follicle development, during which epithelial stem cells proliferate in the hair bulb; later stage (telogen) hairs are absent 18 . Hair bulbs lack internal and external root sheaths in this condition. Taken together, these findings suggest perturbation in the proliferation, survival or differentiation of epithelial stem cell-derived cells residing in hair follicles. Our results implicate SHOC2-mediated signal transduction in this aspect of stem cell biology, which must await the availability of a suitable animal model for precise delineation.
Lastly, as disclosed herein, the human interactome and a network-based statistical method were successfully used to predict a novel gene for human disease. The leading candidate, SHOC2, was a relatively obscure gene that caused no phenotype when mutated in worms, evidence of the strength of this approach. For other projects, one can anticipate that successful candidates will not be deemed this favourably, necessitating resequencing of many low-probability candidate genes. Emerging interactome datasets and improved analytic methods are likely to enhance the predictive power of systems biology.
Materials and Methods
Constructing a Mammalian Protein-Protein Interaction Network from Available Resources
The protein-protein and signalling networks chosen are all literature-based “legacy” direct biochemical mammalian interactions from low-throughput functional experiments extracted manually by expert biologists (literature-curated). Interactions from high-throughput methods, orthologous interactions from lower organisms, or interactions predicted using in silico methods were not included. Only direct biophysical binding or enzymatic interactions were considered, while interactions based on functional association were excluded. The following available protein-protein interaction datasets were used: DIP 19 (http://dip.doe-mbi.ucla.edu/, May 30, 2006); IntAct 20 (ftp://ftp.ebi.ac.uk/pub/databases/intact/current, Jun. 12, 2006); MINT 21 (http://mint.bio.uniroma2.it/mint-old/release/main.php, May 21, 2006); Ma'ayan et al. 22 (http://www.mssm.edu/labs/iyenear/resources, May 21, 2006); BIND 23 (http://www.bind.ca/, Jan. 24, 2006); PDZBase 24 (http://icb.med.cornell.edu/services/pdz/start, Sep. 25, 2006). These datasets were chosen, because components in those networks were annotated with accession codes that permit data consolidation and those datasets were provided freely for analysis and reuse. All interactions from these databases claimed to be direct biochemical interactions determined experimentally, and include the PubMed reference of the research article that describes the experiments used to identify the interactions. Consolidating interactions from the different network databases was accomplished by combining human/mouse/rat gene symbols using the xml version of Swiss Prot (http://www.pir.uniprot.org/database/, on Jun. 21, 2006). The consolidated interactions were stored in a flat file format (http://www.mssm.edu/labs/iyengar/resources/datasets/sig_format.shtml).
Algorithm Used to Generate a List of Novel Ns Candidate Genes.
Problem:
Given a graph G in which a small subset of vertices S, S⊂G, are identified as seed nodes in this case known disease genes that cause NS, find a close to minimum connected subgraph G′ that includes the seed nodes in S while pruning out intermediate nodes and links that are not statistically significant for interacting with the seed list.
Algorithm:
1. Combine available mammalian protein-protein interaction networks using the method described above.
2. Filter the merged network to prune out interactions from publications reporting high-throughput interaction data as described in Berger et al. 10
3. Find all shortest paths 31 of length k 1 between all pairs of vertices in the merged seed list S′∪S″ of all known NS disease genes.
4. Find all edges between intermediate vertices identified in 3. Intermediate vertices, I, are vertices that fall on shortest paths between pairs between all pairs in S′∪S″ such that I⊂G and I {S′∪S″}.
5. Combine all nodes and links found in 3 and 4 to create the subnetwork G′.
6. Rank intermediates base on their links in background network vs. links in subnetwork using a Binomial proportions test as described in Berger et al. 10 .
Subjects and Mutation Analysis.
Genomic DNAs from a cohort of 96 subjects with NS or a phenotype suggestive of this disorder without mutation in previously identified disease genes (PTPN11, SOS1, KRAS, HRAS, RAF1, BRAF, MEK1 and MEK2) were screened for the entire SHOC2 coding region using high-throughput resequencing as previously described. 25 All sequence variants identified were verified by manual inspection of the chromatograms and putative causative mutations were verified using another independent sequencing reaction. SHOC2 was then analyzed in a panel of 410 mutation-negative individuals with NS or a clinically related phenotype with denaturing high-performance liquid chromatography and direct sequencing. 25 DNA from skin fibroblasts, hair bulbs and/or epithelial cells from the oral mucosa was extracted using standard protocols. Samples were collected under Institutional Review Board-approved protocols, with informed consent. Permission was obtained to publish the photographs of subjects shown in FIG. 1 . When available, parental DNAs were sequenced to establish whether identified changes were de novo. Paternity was confirmed using the AmpF/STR Identifier PCR Amplification Kit (Applied Biosystems).
Functional Analyses.
In silico analysis of protein N-myristoylation was performed using Myristoylator (http://www.expasy.org/tools/myristoylator/), TermiNator (http://www.isv.cnrs-gif.fr/terminator3/index.html) and NMT (http.//mendel.imp.ac.at/sat/myristate/index.html) software. The nucleotide substitutions of interest were introduced in V5- and Myc-tagged human SHOC2 cDNA expression constructs by site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit, Stratagene). COS-1, 293-T and Neuro2A cells were maintained in DMEM (GIBCO) supplemented with 10% heat-inactivated FCS (Eueoclone) and antibiotics, and transfected at 60-70% confluency, using Fugene6 (Roche) or Lipofectamine 2000 (Invitrogen). N-myristoylation was evaluated by [ 3 H]myristic acid (30 μCi/ml) incorporation as described elsewhere 26 . Proteins immunoprecipitated with an anti-V5 antibody from cell lysates were separated by SDS-PAGE. Gels were fixed, soaked in Amplify™ (Perkin Elmer) for 30 min, dried under a GelAir drying frame (BioRad), and exposed to X-ray film (Kodak) for two months. Cellular fractionation and ERK phosphorylation assays were performed on Cos-1 cells transiently expressing the V5 tagged SHOC2 wt or SHOC2 S2G using 1 standard protocols 25,27 . Cells were serum starved (16 h) and then stimulated with EGF (30 to 100 ng/ml) for the indicated intervals. In all experiments, a human NMT1 cDNA expression construct (Origene) was co-transfected.
Confocal Laser Scanning Microscopy.
3×10 3 cells were seeded on glass coverslips, transiently transfected, serum starved (16 h) and stimulated with EGF (30 ng/ml, 15 min). Cells were fixed with 3% paraformaldehyde (30 min, 4° C.), permeabilized with 0.5% Triton X-100 (10 min, room temperature), and stained as described in the figure legends. Imaging was performed on a Leica TCS SP2 AOBS apparatus, utilizing excitation spectral laser lines at 405, 488 and 594 nm, tuned with an acousto-optical tunable filter. Image acquisition and processing were conducted by using the Leica Confocal Software (Leica Lasertechnik GmbH). Signals from different fluorescent probes were taken in sequential scanning mode.
Generation of C. elegans Strains and Phenotypic Analysis.
Culture, maintenance and genetic crosses for nematodes were as described 28 . Nematode strains were provided by the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, Minn.). V5-tagged SHOC2 wt and SHOC2 S2G cDNA were subcloned into the heat shock inducible pPD49.83 vector (a gift of Andrew Fire, Stanford University School of Medicine, Stanford, Calif.). A chimeric SHOC2 protein, myr::SHOC2 wt , in which the first seven amino acid residues were substituted by the N-terminal myristoylation signal (MGSCIGK) of src-2 was obtained via PCR amplification and cloned into the pPD49.83 vector. Germline transformation was performed as described 29 . elt-2::GFP (pJM67, a gift from James D. McGhee, University of Calgary, Calgary, Canada), which drives GFP expression in intestinal cells, was used as co-injection marker. At least three independent lines for each construct were tested for the Pv1 phenotype after heat shock. All the lines expressing SHOC2 S2G or myr::SHOC2 wt upon heat shock exhibited a Pv1 phenotype. Only the lines carrying the following transgenes were scored quantitatively at the compound microscope and used for further analyses and crosses: gbEx240[hsp16.2::SHOC2 WT ::V5; pelt-2::GFP], gbEx208a[hsp16.2::SHOC2 S2G ::V5; pelt-2::GFP] and gbEx209[hsp16.2::myr::SHOC2 wt ::V5; pelt-2::GFP]. Genetic crosses were performed according to standard methods. For sur-8(ku167), let-60(n1046), gbEx240 and sur-8(ku167), let-60(n1046), gbEx208a double mutants and let-23(sy1); gbEx240 and let-23(sy1); gbEx208a single mutants the presence of sur-8(ku167), let-60(n1046) and let-23(sy1) mutations was confirmed by sequencing the appropriate region of genomic DNA from each transgenic strain. After each cross, isogenic worms that had lost the transgene were cloned separately and used as controls. N2 and derivative strains were maintained and grown at 20° C. unless otherwise specified. The following mutant alleles were used: sur-8 rf : sur-8(ku167) IV; let-60 gof : let-60(n1046) IV; let-23 rf : let-23(sy1) II. Animals were scored blindly at the dissecting microscope to count the number of eggs in utero after cutting the mother (Eg1), animals that had become bags of worms (Bag) and to check for the presence of multiple ectopic pseudovulvae (Muv). A subset of worms was also scored blindly at the compound microscope for vulva morphology and VPC induction phenotypes.
C. elegans Heat Shock Experiments, Microscopy and Immunocytochemistry.
At different developmental stages, worms carrying the transgenes were subjected to heat shock at 33° C. for 30 min and then kept at 30° C. for 1 h. Synchronized embryos were heat shocked to study the effects of transgene expression on embryonic and early larval development, while synchronized L1/L2 larvae were heat shocked to study the effects on later larval development, movement and fertility. To study VPC induction and vulva morphogenesis, hermaphrodites were heat shocked at early L3 stages and animals were scored for vulval induction at the L4 stage and for Pv1 phenotype at the adult stage. Microscopy observations were performed with a Zeiss Axioskop equipped with epifluorescence and Differential Interference Contrast on live animals anesthetized and mounted on 2% agarose pads containing 10 mM Na-Azide. Images were collected with an Axiocam digital camera. Confocal analyses were performed using a Leica TCS SP2 confocal microscope. For immunocytochemistry analyses, transgenic worms were heat shocked, and after 2 h were fixed with 2% PFA (R.T. 5 min, 1 h on ice). They were processed as reported 30 , and then incubated overnight in a dilution of anti-V5 monoclonal antibody (1:200). After repeated washing (24 h), animals were incubated overnight with Texas-Red conjugated anti-mouse secondary antibody (1:100) (Invitrogen), washed and mounted for observation on microscope slides.
The present invention is also described and demonstrated by way of the above examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.
REFERENCES


1. Resh, M. D. Trafficking and signaling by fatty-acylated and prenylated proteins. Nat Chem Biol 2, 584-90 (2006).
2. Farazi, T. A., Waksman, G. & Gordon, J. I. The biology and enzymology of protein N-myristoylation. J Biol Chem 276, 39501-4 (2001).
3. Selfors, L. M., Schutzman, J. L., Borland, C. Z. & Stern, M. J. soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling. Proc Natl Acad Sci USA 95, 6903-8 (1998).
4. Sieburth, D. S., Sun, Q. & Han, M. SUR-8, a conserved Ras-binding protein with leucine-rich repeats, positively regulates Ras-mediated signaling in C. elegans. Cell 94, 119-30 (1998).
5. Li, W., Han, M & Guan, K. L. The leucine-rich repeat protein SUR-8 enhances MAP kinase activation and forms a complex with Ras and Raf. Genes Dev 14, 895-900 (2000).
6. Rodriguez-Viciana, P., Oses-Prieto, J., Burlingame, A., Fried, M. & McCormick, F. A phosphatase holoenzyme comprised of Shoc2/Sur8 and the catalytic subunit of PP1 functions as an M-Ras effector to modulate Raf activity. Mol Cell 22, 217-30 (2006).
7. Mazzanti, L. et al. Noonan-like syndrome with loose anagen hair: a new syndrome? Am J Med Genet A 118A, 279-86 (2003).
8. Schubbert, S., Shannon, K, & Bollag, G. Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer 7, 295-308 (2007).
9. Tartaglia, M. & Gelb, B. D. Molecular genetics of Noonan syndrome. In Monographs in Human Genetics. Noonan syndrome and related disorders: A matter of deregulated RAS signalling (ed. Zenker, M.) 17, 20-39 (Karger Press, Basel, Switzerland, 2009).
10. Berger, S. I., Posner, J. M. & Ma'ayan, A. Genes2Networks: connecting lists of gene symbols using mammalian protein interactions databases. BMC Bioinformatics 8, 372 (2007).
11. Boutin, J. A. Myristoylation. Cell Signal 9, 15-35 (1997).
12. Tartaglia, M. et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet. 34, 148-50 (2003).
13. Araki, T. et al. Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 10, 849-57 (2004).
14. Loh, M. L. et al. Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 103, 2325-31 (2004).
15. Sternberg, P. W. Vulval development. In Wormbook (ed. The C. elegans Research Community) WormBook, doi/10.1895/wormbook.1.6.1, http://www.wormbook.org (2005).
16. Eisenmann, D. M. & Kim, S. K. Protruding vulva mutants identify novel loci and Wnt signaling factors that function during Caenorhabditis elegans vulva development. Genetics 156, 1097-116 (2000).
17. Kishore, R. S. & Sundaram, M. V. ced-10 Rac and mig-2 function redundantly and act with unc-73 trio to control the orientation of vulval cell divisions and migrations in Caenorhabditis elegans. Dev Biol 241, 339-48 (2002).
18. Tosti, A. et al. Loose anagen hair in a child with Noonan's syndrome. Dermatologica 182, 247-9 (1991).
19. Xenarios, I. et al. The Database of Interacting Proteins: 2001 update. Nucl Acids Res 29, 239-41 (2001).
20. Kerrien, S. et al. IntAct-open source resource for molecular interaction data. Nucl Acids Res 35, D561-D565 (2007).
21. Chatraryamontri, A. et al. MINT: the Molecular INTeraction database. Nucl Acids Res 35, D572-4 (2007).
22. Ma'ayan, A. et al. Formation of regulatory patterns during signal propagation in a Mammalian cellular network. Science 309, 1078-83 (2005).
23. Bader, G. D., Betel, D. & Hogue, C. W. V. BIND: the Biomolecular Interaction Network Database. Nucl Acids Res 31, 248-50 (2003).
24. Beuming, T., Skrabanek, L., Niv, M. Y., Mukherjee, P. & Weinstein, H. PDZBase: a protein-protein interaction database for PDZ-domains. Bioinformatics 21, 827-8 (2005).
25. Tartaglia, M. et al. Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet. 39, 75-9 (2007).
26. Utsumi, T. et al. Amino acid residue penultimate to the amino-terminal Gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation. J Biol Chem 276, 10505-13 (2001).
27. Vachon, L., Costa, T. & Hertz, A. GTPase and adenylate cyclase desensitize at different rates in NG108-15 cells. Mol Pharmacol 31, 159-68 (1987).
28. Sulston, J. E. & Hodgkin, J. Methods. In The Nematode Caenorhabditis elegans (ed. Wood W. B. and the Community of C. elegans Researchers) 587-606 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988).
29. Mello, C. C., Kramer, J. M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans after microinjection of DNA into germline cytoplasm: extrachromosomal maintenance and intergration of transforming sequences. EMBO J 10, 3959-70 (1991).
30. Duerr, J. S. Immunohistochemistry. In WormBook (ed. The C. elegans Research Community,) WormBook, doi/10.1895/wormbook.1.105.1, http://www.wormbook.org (2006).
31. Dijkstra, E. W. A note on two problems in connexion with graphs. Numerische Mathematik 1, 269-271 (1959).