WO2003029422A2 - Noonan syndrome gene - Google Patents

Abstract

Diagnostic and therapeutic applications for Noonan Syndrome are described. The diagnostic and therapeutic applications are based on certain mutations in the protein tyrosine phosphatase gene PTPN11 and its expression product, PTPN11. Also described are nucleotide sequences, amino acid sequences, probes, and primers related to PTPN11 and PTP11 variants, and cells expressing such variants.

Description

Noonan Syndrome Gene

This application claims priority from U.S. Provisional Application Serial No. 60/326,532, filed October 1, 2001, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to diagnostic and therapeutic applications for Noonan Syndrome. In particular, diagnostic and therapeutic applications based on certain mutations in the protein tyrosine phosphatase gene PTPNl 1 or its expression product are contemplated.

BACKGROUND OF THE INVENTION

Noonan syndrome (NS) is an autosomal dominant disorder characterized by dysmorphic facial features, proportionate short stature, and heart disease, i.e., pulmonic stenosis and hypertrophic cardiomyopathy most commonly (Noonan, Am. J. Dis. Child. 1968, 116:373-380; Allanson, J. Med. Genet. 1987, 24:9-13). Webbed neck, chest deformity, cryptorchidism, mental retardation, and bleeding diatheses constitute other frequently associated findings. NS is a relatively common syndrome with an estimated incidence of 1 : 1000 to 1 :2500 live births. At present there is no blood test for diagnosing Noonan syndrome, or any other conclusive genetic test for this disease. The diagnosis depends on recognition of the symptoms by a knowledgeable doctor. However, substantial phenotypic variations, including mild or subtle cases, make the diagnosis difficult. Furthermore, the facial characteristics become less apparent with progressing age, and so the condition sometimes remains undiagnosed. In addition, whereas the disorder may be suspected in utero when fetal ultrasound show excess fetal fluid, such as nuchal edema, there is no reliable test for Noonan syndrome during pregnancy. A number of therapeutic or surgical treatments are available to alleviate various symptoms of Noonan syndrome. Such symptoms include heart defects, undescended testicles, or excessively short stature. However, no therapeutic treatment of the underlying disorder has been proposed so far, absent identification of the causative gene of the Noonan syndrome. Physical mapping of the genetic region involved in Noonan syndrome has been described. A 5-cM region (NSl) on chromosome 12q24.1 has been disclosed, and genetic heterogeneity has also been documented (Jamieson et al., Nat Genet 1994;8:357-360; Brady et al., Eur J Hum Genet 1997 ;5:336-337; Legius et al, Eur J Hum Genet 1998;6:32-37; van Der Burgt, and Brunner, Am J Med Genet 2000;94:46-51). However no specific gene has been identified.

Therefore, there remains a need to identify a specific gene involved in Noonan syndrome. Such identification would immediately provide strategies for diagnosis, in particular early diagnosis, and treatment of this disorder.

SUMMARY OF THE INVENTION

The present invention provides methods of diagnosing and treating Noonan syndrome (NS). By identifying mutations in protein tyrosine phosphatase gene PTPNl 1 in subjects with Noonan syndrome, the inventors provide tools for developing diagnostic and therapeutic applications. Thus, the invention provides a method for diagnosing Noonan syndrome in a subject, which method comprises detecting a mutation in a protein tyrosine phosphatase 11 (PTPNl 1) gene in the subject, wherein the mutation results in increased PTPNl 1 expression or activity as compared to a control. The mutation can be a missense mutation, a deletion, an insertion, and both a deletion and an insertion. Preferably, the mutation is in a coding region of the gene, and results in a mutation in the PTPNl 1 protein. For example, in one embodiment, the mutation in the PTPNl 1 protein is in an src-homology 2 (SH2) domain, including, but not limited to amino acid substitutions selected from the following residues of SEQ ID NO:2: a G to A substitution at position 60; a D to N substitution at position 61; a D to G substitution at position 61; a Y to D substitution at position 62; a Y to C substitution at position 63; an A to S substitution at position 72; an A to G substitution at position 72; an E to D substitution at position 76; and a Q to R substitution at position 79. h another embodiment, the mutation is an amino acid substitution selected from the following residues of SEQ ID NO:2: a T to A substitution at position 42; a G to V substitution at position 60; a T to I substitution at position 73; and an E to D substitution at position 139. In a particular embodiment, the PTPNl 1 protein further comprises a deletion of D at positon 61 of SEQ ID NO:2. Regarding PTPNl 1 gene mutations in the region encoding for an SH2 domain, they may be selected from nucleotide substitutions at the following residues of SEQ ED NO:l: a G to C substitution at position 179; a G to A substitution at position 181; an A to G substitution at position 182; a T to G substitution at position 184; an A to G substitution at position 188; a G to T substitution at position 214; a C to G substitution at position 215; a G to C substitution at position 228; and an A to G substitution at position 236. Alternatively, the mutation may be selected from an A to G substitution at position 124; a G to T substitution at position 179 of SEQ ED NO:l; a C to T substitution at position 218; a G to C substitution at position 417; and a G to T substitution at position 417. In a particular embodiment, the PTPNl 1 gene further comprises a deletion of positions 180-182 of SEQ ID NO: 1.

In a further embodiment, the mutation in the PTPNl 1 protein is in a linker domain connecting a first SH2 domain to a second SH2 domain (See Figure 1). For example, the mutation can be a D to A amino acid substitution at position 106 of SEQ ED NO:2, preferably corresponding to an A to C substitution at position 317 of SEQ ED NO: 1.

In yet a further embodiment, the mutation in the PTPNl 1 protein is in a protein tyrosine phosphatase (PTP) domain. For example, the mutation can be an amino acid substitution selected from the following residues of SEQ ID NO:2: a Y to C substitution at position 279; an I to V substitution at position 282; an F to L substitution at position 285; an F to S substitution at position 285; an N to D substitution at position 308; an N to S substitution at position 308; an I to V substitution at position 309; an R to K substitution at position 501 of SEQ ED NO:2; and an M to V substitution at position 504. Alternatively, the mutation can be an amino acid substitution selected from the following amino acid residues of SEQ ED NO:2: a G to S substitution at position 268; an N to T substitution at position 308; a P to S substitution at position 491 ; and an S to L substitution at position 502.

Regarding PTPNl 1 gene mutations in the region encoding for a PTP domain, they may be selected from nucleotide substitutions at the following residues of SEQ ED NO:l: an A to G substitution at position 836; an A to G substitution at position 844; a T to C substitution at position 853; a T to C substitution at position 854; an A to G substitution at position 922; an A to G substitution at position 923; an A to G substitution at position 925; a G to A substitution at position 1502; and an A to G substitution at position 1510. Alternatively, the gene mutations may be selected from a G to A substitution at position 802; an A to C substitution at position 923; a C to T substitution at position 1471; and a C to T substitution at position 1505 of SEQ ED NO:l.

The invention also provides for a kit for diagnosing Noonan syndrome, comprising an oligonucleotide that specifically hybridizes to or adjacent to a site of mutation of a PTPNl 1 gene that results in increased activity of a PTPNl 1 protein encoded by the gene, and instructions for use. The site of mutation may, for example, comprise a nucleotide selected from the group consisting of nucleotides 179, 181, 182, 184, 188, 214, 215, 228, 236, 317, 836, 844, 853, 854, 922, 923, 925, 1502, and 1510 of SEQ TD NO:l. h another embodiment, the site of mutation comprises a nucleotide selected from the group consisting of nucleotides 124, 180, 218, 417, 802, 1403, 1471, and 1505 of SEQ ED NO:l. hi one preferred embodiment, the kit comprises at least one probe comprising the site of mutation. In another preferred embodiment, the kit comprises a first oligonucleotide primer comprising at least 15 consecutive nucleotides of SEQ ED NO:33, and a second oligonucleotide primer comprising at least 15 consecutive nucleotides of a sequence complementary to SEQ ED NO:33. Such primer pairs may be selected from, for example, a first primer comprising a nucleotide sequence selected from the group consisting of SEQ ED NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31, and a second primer selected from the group consisting of SEQ TD NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32.

The invention further provides for a kit for diagnosing Noonan syndrome, comprising an antibody that specifically recognizes a mutation in a PTPNl 1 protein, and instructions for use. Optionally, the mutation results in an increased activity as compared to a PTPNl 1 protein having the amino acid sequence of a wild-type PTPNl 1, such as that of SEQ TD NO:2. The mutation may be in an SH2 domain, and is preferably selected from the following amino acid substitutions in SEQ ED NO:2: a G to A substitution at position 60; a D to N substitution at position 61; a D to G substitution at position 61; a Y to D substitution at position 62; a Y to C substitution at position 63; an A to S substitution at position 72; an A to G substitution at position 72; an E to D substitution at position 76; and a Q to R substitution at position 79. Alternatively, the mutation may be selected from a T to A substitution at position 42; a G to V substitution at position 60 and a deletion of position 61 of SEQ ED NO:2 a T to I substitution at position 73; and an E to D substitution at position 139 of SEQ TD NO:2.

The mutation may also be in a linker domain connecting a first SH2 domain to a second SH2 domain. For example, the mutation can be a D to A substitution at position 106 of SEQ TD NO:2.

In addition, the mutation can be in a PTP domain, and is preferably selected from the following amino acid substitutions in SEQ ED NO:2: an Y to C substitution at position 279; an I to V substitution at position 282; an F to L substitution at position 285; an F to S substitution at position 285; an N to D substitution at position 308; an N to S substitution at position 308; an I to V substitution at position 309; an R to K substitution at position 501; and an M to V substitution at position 504. Alternatively, the mutation can be selected from the following: a G to S substitution at position 268; an N to T substitution at position 308; a P to S substitution at position 491; and an S to L substitution at position 502 of SEQ ED NO:2.

The invention also provides for a method for diagnosing Noonan syndrome in a subject, which method comprises assessing the level of expression or activity of a PTPNl 1 protein in the test subject and comparing it to the level of expression or activity in a control subject, wherein an increased expression or basal activity of the PTPNl 1 protein in the test subject compared to the control subject is indicative of Noonan syndrome. The level of expression may, for example, be assessed by determining the amount of mRNA that encodes the PTPNl 1 protein in a biological sample or by deteπnining the concentration of PTPNl 1 protein in a biological sample. The level of activity may, for example, be assessed by determining the level of PTPNl 1 phosphatase activity in the test subject. The invention further provides for a method for treating Noonan syndrome in a patient, which method comprises administering to the patient in need of such treatment an effective amount of an agent that modulates the expression or activity of a PTPNl 1 protein, in association with a pharmaceutically acceptable carrier. Preferably, although not necessarily, the PTPNl 1 protein comprises the amino acid sequence of SEQ ED NO:2.

Ln one embodiment, the agent is a PTPNl 1 antisense nucleic acid, preferably an antisense nucleic acid hybridizing to a segment of SEQ ED NO:l comprising at least one nucleotide substitution selected from a G to C substitution at position 179; a G to A substitution at position 181; an A to G substitution at position 182; a T to G substitution at position 184; an A to G substitution at position 188; a G to T substitution at position 214; a C to G substitution at position 215; an G to C substitution at position 228; an A to G substitution at position 236 of SEQ ED NO:l; and complementary segments thereof. Alternatively, the antisense nucleic acid can hybridize to a segment of SEQ ED NO:l comprising at least one nucleotide substitution selected from an A to G substitution at position 124; a G to T substitution at position 179; a C to T substitution at position 218; a G to C substitution at position 417; a G to T substitution at position 417 of SEQ ED NO:l; and complementary segments thereof. Ln a particular embodiment, the segment of the PTPNl 1 gene further comprises a deletion of positions 180-182 of SEQ ED NO:l. h a further embodiment, the antisense nucleic acid hybridizes to a segment of SEQ ED NO:l comprising at least one nucleotide substitution selected from: an A to G substitution at position 836; an A to G substitution at position 844; a T to C substitution at position 853; a T to C substitution at position 854; an A to G substitution at position 922; an A to G substitution at position 923; an A to G substitution at position 925; a G to A substitution at position 1502; an A to G substitution at position 1510 of SEQ ED NO:l; and complementary sequences thereof. Alternatively, the antisense nucleic acid hybridizes to a segment of SEQ ED NO:l comprising at least one nucleotide substitution selected from an G to A substitution at position 802; an A to C substitution at position 923; a C to T substitution at position 1471; and a C to T substitution at position 1505 of SEQ ED NO:l; and complementary segments thereof.

In a specific embodiment, the agent inhibits PTPNl 1 activity by blocking a PTP domain. For example, the agent can be an anti-PTPNl 1 inhibitory antibody. Such an antibody could specifically recognize a PTPNl 1 amino acid sequence comprising mutation selected from the following residues of SEQ ED NO:2: a G to A substitution at position 60; a D to N substitution at position 61; a D to G substitution at position 61; a Y to D substitution at position 62; a Y to C substitution at position 63; an A to S substitution at position 72; an A to G substitution at position 72; an E to D substitution at position 76; a Q to R substitution at position 79; a D to A substitution at position 106; an Y to C substitution at position 279; an I to V substitution at position 282; an F to L substitution at position 285; an F to S substitution at position 285; an N to D substitution at position 308; an N to S substitution at position 308; an I to V substitution at position 309; an R to K substitution at position 501 ; and an M to V substitution at position 504. Alternatively, the antibody may specifically recognize a PTPNl 1 mutation in SEQ ED NO:2 selected from the following: a T to A substitution at position 42; a G toV substitution at position 60 and a deletion of position 61; a T to I substitution at position 73; an E to D substitution at position 139; a G to S substitution at position 268 of SEQ ID NO:2; an N to T substitution at position 308; an P to S substitution at position 491; and an S to L substitution at position 502.

The invention also provides for an isolated PTPNl 1 variant comprising a mutation resulting in increased level of PTPNl 1 activity. In particular embodiments, the isolated PTPNl 1 variant comprises an amino acid substitution in an N-SH2 domain, C-SH2 domain, or PTP domain, or in a region between an N-SH2 and a C-SH2 domain. The amino acid substitution may be selected from the following group, referring to residues in SEQ ED NO:2: a G to A substitution at position 60; a D to N substitution at position 61; a D to G substitution at position 61; a Y to D substitution at position 62; a Y to C substitution at position 63; an A to S substitution at position 72; an A to G substitution at position 72; an E to D substitution at position 76; a Q to R substitution at position 79; a D to A substitution at position 106; an Y to C substitution at position 279; an I to V substitution at position 282; an F to L substitution at position 285; an F to S substitution at position 285; an N to D substitution at position 308; an N to S substitution at position 308; an I to V substitution at position 309; an R to K substitution at position 501; and an M to V substitution at position 504. Alternatively, the amino acid mutation can be selected from the following: a T to A substitution at position 42; a G toV substitution at position 60 and a deletion of position 61; a T to I substitution at position 73 of SEQ ED NO:2; an E to D substitution at position 139; a G to S substitution at position 268; an N to T substitution at position 308; an P to S substitution at position 491; and an S to L substitution at position 502 of SEQ ED NO:2.

The invention further provides for an isolated cell comprising a vector, which vector comprises a nucleic acid encoding any PTPNl 1 variant described above, the nucleic acid operatively associated with an expression control sequence. The cell can be, for example, a prokaryotic cell or an eukaryotic cell.

The invention also provides for an isolated nucleic acid encoding any of the PTPNl 1 variants described above, as well as isolated oligonucleotides which specifically hybridizes to such nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic drawing showing PTPNl 1 gene organization and domain structure. The numbered, filled boxes at the top indicate the coding exons; the positions of the ATG and TGA codons are shown. The functional domains of the PTPNl 1 protein, consisting of two tandemly arranged src-homology 2(SH2) domains at the N-terminus (N-SH2 and C-SH2) followed by a protein tyrosine phosphatase (PTP) domain, are shown below. The numbers below that cartoon indicate the amino acid boundaries of those domains. Figure 2 shows the distribution of PTPNl 1 (SHP-2) mutations and their relative prevalence in Noonan syndrome.

Figures 3A and 3B show sequence analysis of amplified DNA containing the PTPNl 1 exon 3 and flanking intronic boundaries, which revealed heterozygous changes in the NS-C and NS-L families. Figure 3A shows a G-to-T transversion at nt (nucleotide) 214 in an affected individual from NS-C. Figure 3B shows an A-to-G transition at nt 236 in an individual from NS-L. Wild-type exonic sequences and the amino acid residues for which they encode are shown below the electrophoregrams. Bases that were mutated are shown in bold with arrows indicating the position of the heterozygous sequences. Figures 4 A and 4B show segregation of the G214T (Figure 4 A) and

A236G (Figure 4B) mutations in NS-C and NS-L families, respectively. Family trees for these two families are shown above. A single Bgϊl site was present in the wild- type amplified product so that this digestion resulted in 164- and 374-bp (base pair) products. The G214T change obliterated that BgR site so that the heterozygotes have undigested 538-bp product as well. The A236G mutation introduced a novel BsaΨI restriction site, resulting in 177- and 361-bp products, whereas wild type alleles remained undigested at 538 bp.

Figure 5 shows a restriction analysis of PTPNl 1 PCR product containing exon 8, showing segregation of the 922A-»G (A922G) mutation in the original large family with NS that shows linkage to 12q24 (Jamieson et al. Nat Genet 1994;8:357-360). The family tree is shown above. The mutation introduces an EcoRV restriction site, resulting in 246- and 366-bp products, whereas the wild-type allele remains undigested (612 bp).

Figure 6 shows DHPLC elution profiles of PTPNl 1 exon 8 PCR products, showing segregation of the 923 A— »G (A923G) change in a family inheriting Noonan-like/multiple giant-cell lesion syndrome (Bertola et al., Am J Med Genet 2001;98:230-234). A single peak characterizes the wild-type profile, and a variant profile, characterized by two peaks, is observed in all affected family members. The family tree is shown on the left. The gray-shaded symbol indicates an indeterminate phenotype.

Figure 7 shows the increased phosphatase activity observed in the mutant PTPNl 1 proteins. Wild type and mutant PTPNl 1-N5 proteins were immunoprecipitated using anti-N5 and the phosphatase activity measured with the malachite green assay. The absorbance at 620 nm is indicated on the y axis. A phosphate standard curve was performed, documenting linearity in this range of absorbance. The white bars indicate the phosphatase activities of wild type and mutant PTPΝ11 proteins isolated from starved, unstimulated COS-7 cells. The black bars indicate the phosphatase activities of wild type and mutant PTPΝ11 protein isolated from COS 7 cell that were stimulated for 5 min with EGF. See Example 3.

Figures 8A and 8B describe the results of immunoblotting experiments comparing docking activity of PTPΝ11 and PTPΝ11 variant N5- constructs to Gabl (docking partner) under basal and activated conditions (i.e., with or without EGF-stimulation), showing increased docking function of PTPΝ11 variants. See Example 3. A. Comparison of the quantities of PTPΝ11 detected revealed that there was almost none docked to Gabl prior to EGF stimulation and modest amounts thereafter. In contrast, the A72S PTPΝ11 mutant co-isolated with Gabl without EGF stimulation and was present in much larger amounts compared to wild type PTPNl 1 after EGF stimulation. B. For the wild type, there was very little detectable Gabl co-isolated prior to EGF stimulation, a brisk increase peaking around 5 min and then at taper nearly back to baseline by 60 min. In contract, high amounts of Gabl were still co-localizing with N308D PTPNl 1 at 60 min.

Figure 9 shows a prolonged increase in ERK2 kinase activity after EGF stimulation of COS-7 cells expressing mutant PTPNl 1. Cells were co- transfected with a construct for HA-ERK2 and that protein was immunoprecipitated from cell lysates from 0, 15, 30, and 60 min after EGF stimulation. Kinase activity was assessed using myelin basic protein (MBP). The bottom panel indicates roughly equivalent amounts of HA-ERK2 in all conditions. There was no detectable ERK2 kinase activity prior to EGF stimulation for wild type PTPNl 1, but a small amount with N308D mutant (even though there was relatively less HA-ERK2 for this condition). For the wild type, the kinase activity increased briskly at 5 min and then tapered off dramatically by 60 min. For the N308D, there was a similar increase in ERK2 kinase activity at 5 min but the activity remained modestly elevated even at 60 min. See Example 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is, in part, based on the identification of mutations in the protein tyrosine phosphatase gene, PTPNl 1, that are causative for, or associate closely with, Noonan Syndrome (NS). In particular, the invention provides mutant PTPNl 1 coding and non-coding nucleotide sequences associated with NS. The invention further provides PTPNl 1 polypeptides that are encoded by such variant nucleic acids and/or comprise one or more amino acid residue substitutions, insertions or deletions. Preferably, the PTPNl 1 or PTPNl 1 variants are characterized by an increased PTPNl 1 activity, i.e., gain-of-function in PTPNl 1 activity; or by higher PTPNl 1 expression levels, as compared to controls.

The invention also provides antibodies that specifically bind to these variant PTPNl 1 polypeptides, as well as nucleic acids which may be used in the methods of the invention to detect a variant PTPNl 1 nucleic acid or PTPNl 1 gene. For example, in one embodiment, the invention provides oligonucleotides sequences which may be used, e.g., to detect a mutation in a PTPNl 1 gene, or to amplify a PTPNl 1 nucleic acid (for example, a specific locus on a PTPNl 1 gene) having or suspected of having a mutation that correlates to NS.

Methods are also provided, as part of the present invention, which use the nucleic acids, polypeptides and antibodies described herein to diagnose or treat NS. For example, the invention provides methods to evaluate individuals for NS by detecting a variant PTPNl 1 nucleic acid or polypeptide, such as one of the variants described herein, that statistically correlates to NS. The invention also provides therapeutic methods for treating NS by administering a compound that modulates (e.g., enhances or inhibits) the expression or activity of either a PTPNl 1 nucleic acid (e.g., a PTPNl 1 gene) or a PTPNl 1 gene product (e.g., a PTPNl 1 polypeptide). In one preferred embodiment, the compound modulates the expression or activity of a variant PTPNl 1 nucleic acid or expression product, such as one of the variants described herein.

PTPNl 1 was considered as a candidate gene for Noonan syndrome because it mapped to chromosome 12q24.1 between D12S84 and D12S79. This region has previously been identified to be associated with Noonan syndrome. In addition, its protein product occupies a critical role in several intracellular signal transduction pathways controlling diverse developmental processes, including cardiac semilunar valvulogenesis (Dechert, et al, Hum. Genet., 1995, 96: 609-615; Feng, Exp. Cell Res., 1999, 253: 47-54; Chen, et al, Nature Genet., 2000, 24:296-299).

Briefly, as described in the Examples, the PTPNl 1 gene organization and intron boundary sequence were established using cDNA (Genbank Accession Nos. NM_002834; nucleotide and amino acid sequences represented herein as SEQ ED NOS:l and 2, respectively) and genomic sequences (GenBank Accession No. NT_009770, residues 3,000,001-3,300,000 of which represented herein as SEQ ED NO:33; Bacterial Artificial Chromosome (BAC) clone RP3-329E11). Example 1 describes mutation screening with two moderate-sized families, in which the NS phenotype co-segregated with haplotypes defined by D12S84, D12S105, D12S354 and D12S2070. Bi-directional sequencing of the fifteen PTPNl 1 exons and their intron boundaries for the two families revealed mutations in one exon, predicting a substitution in the N-SH2 domain. These sequence changes were confirmed in all affected individuals in the families, but was absent in unaffected family members and controls. Example 2 describes the molecular consequences of the mutations. Example 3 describes analysis of the protein mutants. Example 4 describes the identification of additional mutations and further characterization of the role of the mutations in Noonans syndrome. Taken together, these findings established PTPNl 1 as an NS disease gene.

Definitions a) Noonan syndrome:

As used herein the term "Noonan syndrome" (NS) encompasses all forms of the disorder as described under the accession No. MEM 163950 in the Online Mendelian Inheritance in Man (OMEM) at World-Wide Web Address www.ncbi.nlm.nih.gov/Omim, (as accessed in September, 2001), as well as disorders similar or related to NS. Such disorders include, but are not limited to, the Watson (MM 193520) and LEOPARD (MEM 151100) Syndromes, essentially indistinguishable from NS (Mendez and Opitz, Am J Med Genet 1985;21:493-506); male Turner and female pseudo-Turner Syndrome, as well as Turner phenotype with normal karyotype (see MEM 163950); Noonan syndrome with multiple giant-cell lesions (MIM 163955; Tartaglia et al., Am J Hum Genet 2002;70:1555-1563) and/or Noonan syndrome with multiple cafe-au-lait spots (also known as LEOPARD syndrome, MM 151100; Digilio et al, Am J Hum Genet 2002;71:389-394; Legius et al, J Med Genet 2002;39:571-574); valvular sclerosis (Snellen et al, Circulation 1968;38(1 Suρpl):93-101); and idiopathic short stature (Attie KM, Curr Opin Pediatr 2000;12:400-404).

NS encompasses familial or sporadic forms, including NSl, whose locus has been identified on chromosome 12. The present invention takes into consideration, however, that NS and its related disorders are genetically heterogeneous, but share phenotypical features. The features of NS have been well described and a clinical scoring system devised. See, Mendez and Opitz, Am J Med Genet 1985;21:493-506; Noonan, Clin Pediatr (Phila) 1994;33:548-555; Sharland et al., Arch Dis Child 1992;67:178-183; Duncan et al., Am J Med Genet 1981;10:37- 50). The subject to whom the diagnostic or therapeutic applications of the invention are directed may be any human or animal, more particularly a mammal, preferably a primate or a rodent, but including, without limitation, monkeys, dogs, cats, horses, cows, pigs, sheep, goats, rabbits, guinea pigs, hamsters, mice and rats. The subject may be of any age, e.g., an adult, a child, an infant. Prenatal diagnostics and therapeutics interventions are also encompassed. b) PTPNl 1:

PTPNl 1, also known as SHP-2, Syp, SHPTP2, PTP2C, PTP1D and BPTP3, is a member of the family of non-membrane tyrosine phosphatases and is ubiquitously expressed in all tissues examined, with higher levels of expression in the heart and the brain (Ahmad et al, Proc Natl Acad Sci USA 1993;90:2197-2201; Bastien et al, Biochem Biophys Res Commun 1993 ;196: 124-133; Freeman et al, Proc Natl Acad Sci USA, 1992;89:11239-11243). The function of the SH2 domain is to specifically recognize the phosphorylated state of tyrosine residues, thereby allowing PTPNl 1 to localize to tyrosine-phosphorylated sites.

The genomic sequence of PTPNl 1, is organized as follows (residues referring to SEQ ED NO:33): Exon 1, residues 123211-123604; Exon 2, residues 136831-136953; Exon 3, residues 194431-194625; Exon 4, residues 197308-197500; Exon 5, residues 198677-198793; Exon 6, residues 200063-200176; Exon 7, residues 217057-217153; Exon 8, residues 221764-221843; Exon 9, residues 221970-222128; Exon 10, residues 226187-226318; Exon 11, residues 230588-230742; Exon 12, residues 232556-232623; Exon 13, residues 233137-233288; Exon 14, residues 246257-246369; Exon 15, 248808-248909; Exon 16, residues 249938-250510. The adenine nucleotide of the start codon "ATG" is located at residue No. 123591 of SEQ ED NO:33. A partial genomic sequence is also provided by GenBank Accession No. AC004086.

In the context of the present invention, the PTPNl 1 gene encompasses a gene of human origin, comprising a coding nucleotide sequence set forth in SEQ ED NO:l, or homologs, including allelic variants and orthologs. The PTPNl 1 protein encompasses a PTPNl 1 protein of human origin having the amino acid sequence set forth in SEQ ID NO:2, or homologs, including orthologs thereof. As used herein, the term "PTPN11" in italicized form refers to a nucleotide sequence (genomic, cDNA, etc.), whereas the non-italicized form refers to a peptide or protein sequence. Figure 1 shows the organization of the PTPNl 1 gene and the functional domains of the PTPNl 1 protein. The PTPNl 1 protein comprises two SH2 (src-homology 2) domains, one from amino acid 3 to amino acid 104, the other from amino acid 112 to amino acid 216, and one PTP (protein tyrosine phosphatase) domain, from amino acid 221 to amino acid 524. "PTPNl 1 variants" nucleic acids are PTPN11 genomic DNA, cDNA, or mRNA comprising at least one mutation, preferably a nucleotide substitution. The nucleotide substitution may be in a coding or non-coding region. Preferred PTPNl 1 variants are those resulting in the expression of higher levels of PTPNl 1 as compared to a control, and those encoding PTPNl 1 variants characterized by increased PTPNl 1 activity (i.e., "gain-of function variants").

"PTPNl 1 variants" are PTPNl 1 proteins or polypeptides comprising at least one mutation. The PTPNl 1 variants can be function-conservative variants, including gain-of- function-variants, i.e., variants capable of increased PTPNl 1 activity, such as higher tyrosine phosphatase activity. The increase in PTPNl 1 activity includes, for example, increased phosphatase activity, prolonged activity of PTPNl 1, and a higher proportion of PTPNl 1 remaining in an active state (see below). This may be assessed either by direct measurement of PTPNl 1 activity or by measuring the activity of components regulated by PTPNl 1 activity (see, Example 4). Preferred mutations are amino acid substitutions and deletions, in particular those described in Figure 2 and Table 1.

PTPNl 1 participates in signaling cascades elicited by a number of growth factors, cytokines and hormones (Feng, Exp. Cell Res. 1999;253:47-54; Stein- Gerlach et al, hit. J. Biochem. Cell. Biol. 1998;30:559-566; Tamir, et al, Curr. Opin. Immunol., 2000;12:307-315.) As such, PTPNl 1 plays a role in modulating cellular proliferation, differentiation and migration. Following ligand-induced receptor activation, PTPNl 1 is recruited through its SH2 domains directly by the receptor or indirectly via docking proteins. The C-terminal tail of PTPNl 1 also has tyrosine residues that can become phosphorylated, providing SH2 binding sites for other proteins. Thus, PTPNl 1 may act as a phosphatase and as an adapter molecule with docking function, both functions appearing to be relevant in signal transduction. Depending on the specific signaling pathway, PTPNl 1 can act as either a positive or negative regulator of ERK, Jnk kinase, Jak/STAT, and NF-κB cascades (Saxton, et al, EMBO J.,1997;16:2352-2364; Shi et al, J. Biol. Chem., 1998;273:4904-4908; You et al, Mol. Cell. Biol, 1999;19:2416-2424; Maroun et al, Mol. Cell. Biol., 2000;20:8513-8525; You et al, J. Exp. Med., 2001;193:101-110; and Van Vactor et al., Curr Opin Genetics Development 1998;8:112-126). These various roles of PTPNl 1, as described herein, are also referred to as "functions" or "activities" of the protein.

An "increased activity" of PTPNl 1 in a test subject or a biological sample refers to a higher total PTPNl 1 activity in the test subject or biological sample in comparison with a control, e.g., a healthy subject or a standard sample. Preferably, although not necessarily, the activity is at least 10%, more preferably at least 50%, even more preferably at least 100%, and still more preferably at least 150% higher in the test subject or sample than in the control. The increased activity may results from increased basal PTPNl 1 activity, prolonged stimulation of a downstream component (e.g., ERK2) of a PTPNl 1 -associated pathway, and a higher PTPNl 1 expression level. (See Examples).

Basal level of PTPNl 1 activity is dependent on the conformation of the protein. Wild-type PTPNl 1 exists in an inactive (I) or an active (A) conformation, with the N-SH2 domain acting as a molecular switch. In the I state, N- SH2 assumes a conformation that blocks the PTP active site and disrupts its own, separate phosphopeptide-binding cleft. On binding of phosphopeptide, the N-SH2 domain assumes the A conformation that disrupts its PTP recognition surface. Without being bound to any specific theory, it is believed that mutations in PTPNl 1 observed in Noonan syndrome could result in destabilization of the I state, favoring the A state.

A higher expression level of wild-type or variant PTPNl 1 may result from, for example, a mutation in a non-coding region of a PTPNl 1 gene or a mutation in a coding or non-coding gene involved in PTPNl 1 transcription or translation. The expression level of PTPNl 1 can be determined, for example, comparing PTPNl 1 mRNA or level of PTPNl 1 protein in a test subject as compared to a control. c) Molecular Biology Terms:

In accordance with the present invention 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, New York (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 & SJ. 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 (ERL 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). The terms "polypeptide" and "protein" may be used herein interchangeably to refer to the gene product (or corresponding synthetic product) of a PTPNl 1 gene. The term "protein" may also refer specifically to the polypeptide as expressed in cells.

A "PTPNl 1 gene" is used herein to refer to a portion of a DNA molecule that includes a PTPNl 1 polypeptide coding sequence operatively associated with expression control sequences. Thus, a gene includes both transcribed and untranscribed regions. The transcribed region may include introns, which are spliced out of the mRNA, and 5'- and 3 '-untranslated (UTR) sequences along with protein coding sequences. In one embodiment, the gene can be a genomic or partial genomic sequence, in that it contains one or more introns. In another embodiment, the term gene may refer to a cDNA molecule (i.e., the coding sequence lacking introns). In yet another embodiment, the term gene may refer to expression control sequences, such as the promoter or the enhancer sequence.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

"Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.

"Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide.

Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99%o as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A "variant" also includes a polypeptide or enzyme which has at least 60 %> amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%), and still more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared. A particular variant is a "gain-of-function" variant, meaning a polypeptide variant in which the change of at least one given amino acid residue in a protein or enzyme improves a specific function of the polypeptide, including, but not limited to, protein activity. The change in amino acid residue can be replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like), or different properties.

As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987,Cell 50:667). Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs at conserved positions.

Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin. In a specific embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 80%>, and most preferably at least about 90 or at least 95%) of the nucleotides match over the defined length of the DNA sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc. An example of such a sequence is an allelic or species variant of the PTPNl 1 gene. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80% of the amino acids are identical, or greater than about 90%> or 95% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of the programs described above (BLAST, FASTA, etc.).

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al.). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T_m (melting temperature) of 55°C, can be used, e.g., 5xSSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T_m, e.g., 40%> formamide, with 5x or 6x SCC. High stringency hybridization conditions correspond to the highest T_m, e.g., 50%> formamide, 5x or 6x SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7- 11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides. In a specific embodiment, the term "standard hybridization conditions" refers to a T_m of 55°C, and utilizes conditions as set forth above. Ln a preferred embodiment, the T_m is 60°C; in a more preferred embodiment, the T_m is 65°C. In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68°C in 0.2xSSC, at 42°C in 50% formamide, 4xSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.

The terms "mutant" and "mutation" mean any detectable change in genetic material, e.g., DNA, or any process, mechanism, or result of such a change. When compared to a control material, such change may be referred to as an "abnormality". This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g. protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant. "Amplification" of DNA as used herein encompasses the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki et al, Science 1988, 239:487. "Sequencing" of a nucleic acid includes chemical or enzymatic sequencing. "Chemical sequencing" of DNA denotes methods such as that of Maxam and Gilbert (Maxam-Gilbert sequencing, Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 1977 74:560), in which DNA is randomly cleaved using individual base-specific reactions. "Enzymatic sequencing" of DNA denotes methods such as that of Sanger (Sanger et al, Proc. Natl. Acad. Sci. USA 1977, 74:5463), in which a single-stranded DNA is copied and randomly terminated using DNA polymerase, including variations thereof, which are well-known in the art. Preferably, oligonucleotide sequencing is conducted using automatic, computerized equipment in a high-throughput setting, for example, microarray technology, as described herein. Such high-throughput equipment are commercially available, and techniques well known in the art.

The term "polymorphism" refers, generally, to the coexistence of more than one form of a gene (e.g., more than one allele) within a population of individuals. The different alleles may differ at one or more positions of their nucleic acid sequences, which are referred to herein as "polymorphic locuses". When used herein to describe polypeptides that are encoded by different alleles of a gene, the term "polymorphic locus" also refers to the positions in an amino acid sequence that differ among variant polypeptides encoded by different alleles. Polymorphisms include "single nucleotide polymorphisms" (SNPs), referring to a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. Typically, the polymorphic site of an SNP is flanked by highly conserved sequences (e.g., sequences that vary in less than 1/100 and, more preferably, in less than 1/1000 individuals in a population). The polymorphic locus of an SNP may be a single base deletion, a single base insertion, or a single base substitution. Single base substitutions are particularly preferred.

As used herein, "sequence-specific oligonucleotides" refers to related sets of oligonucleotides that can be used to detect variations or mutations in the PTPNl 1 gene.

A "probe" refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target region due to complementarity of at least one sequence in the probe with a sequence in the target protein.

As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with ³²P -nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of PTPNl 1, or to detect the presence of nucleic acids encoding PTPNl 1. In a further embodiment, an oligonucleotide of the invention can form a triple helix with a PTPNl 1 DNA molecule. In still another embodiment, 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. Specific non-limiting examples of synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH₂-NH-O-CH₂, CH₂-N(CH)₃-O-CH₂, CH₂-O-N(CH)₃-CH₂, CH₂-N(CH)₃-N(CH)₃-CH₂ and O-N(CH)₃-CH₂-CH₂ backbones (where the phosphodiester is O-PO₂-O-CH₂). US Patent No. 5,677,437 describes heteroaromatic olignucleoside linkages. Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U.S. Patents No. 5,792,844 and No. 5,783,682). US Patent No. 5,637,684 describes phosphoramidate and phosphorothioamidate ohgomeric compounds. Also envisioned are oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al., Science 254:1497, 1991). Other synthetic oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH₃, F, OCN, O(CH₂)„NH₂ or O(CH₂)_nCH₃ where n is from 1 to about 10; to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-; S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃ ; SO₂CH₃; ONO₂;NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the phaimacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group. Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine, such as inosine, may be used in an oligonucleotide molecule. The present invention provides antisense nucleic acids (including ribozymes), which may be used to inhibit expression of PTPNl 1. An "antisense nucleic acid" is a single stranded nucleic acid molecule which, on hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid. As presently used, "antisense" broadly includes RNA-RNA interactions, RNA-DNA interactions, ribozymes and RNase-H mediated arrest. Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent No. 5,814,500; U.S. Patent No. 5,811,234), or alternatively they can be prepared synthetically (e.g., U.S. Patent No. 5,780,607). Synthetic oligonucleotides are suitable for antisense use.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Nectors include plasmids, phages, viruses, etc.

The term "linkage" refers to the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. Linkage may be measured, e.g., by the percent recombination between two genes, alleles, loci or genetic markers.

Expression of PTPΝ11 Polypeptides

A nucleotide sequence coding for PTPΝ11, for an antigenic fragment, derivative or analog of PTPΝ11, of for a functionally active derivative of PTPΝ11 (including a chimeric protein) may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Thus, a nucleic acid encoding a PTPNl 1 polypeptide of the invention can be operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. Such vectors can be used to express functional or functionally inactivated PTPNl 1 polypeptides. In particular, the PTPNl 1 nucleic acids which may be cloned and expressed according to these methods include, not only wild-type PTPNl 1 nucleic acids, but also mutant or variant PTPNl 1 nucleic acids. These include, for example, a PTPNl 1 nucleic acid having one or more of the mutations or polymorphisms set forth in Tables 1 and 6. In addition, nucleic acids that encode a variant PTPNl 1 polypeptide, for example a variant PTPNl 1 polypeptide comprising one or more of the amino acid substitutions listed in Table 1 may be cloned and expressed according to the methods described here.

The necessary transcriptional and translational signals can be provided on a recombinant expression vector. Potential host- vector systems include but are not limited to mammalian cell systems transfected with expression plasmids or infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, herpes virus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. Expression of a PTPNl 1 protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control PTPNl 1 gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, Nature 1981, 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 1980, 22:787-797), the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A. 1981, 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 1982, 296:39 42); prokaryotic expression vectors such as the b-lactamase promoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 1978, 75:3727-3731), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. 1983, 80:21-25, 1983); see also "Useful proteins from recombinant bacteria" in Scientific American 1980, 242:74-94. Still other useful promoter elements which may be used include promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and transcriptional control regions that exhibit hematopoietic tissue specificity, in particular: beta-globin gene control region which is active in myeloid cells (Mogram et al., Nature 1985, 315:338-340; Kollias et al., Cell 1986, 46:89-94), hematopoietic stem cell differentiation factor promoters, erythropoietin receptor promoter (Maouche et al., Blood 1991, 15:2557), etc.

Soluble forms of the protein can be obtained by collecting culture fluid, or solubilizing-inclusion bodies, e.g., by treatment with detergent, and if desired sonication or other mechanical processes, as described above. The solubilized or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2 dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al., Gene 1988, 67:31-40), pCR2.1 and pcDNA 3.1+ (Invitrogen, Carlsbad, California), pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like. Preferred vectors are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism. Thus, a gene encoding a functional or mutant PTPNl 1 protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995. Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures (see below), as well as in vitro expression, are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques 1992, 7:980-990). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or can be rendered nonfunctional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Ba r virus (EBV), adenovirus, adeno-associated virus (AAV), baculovirus, and the like. RNA viral vectors include, for example, retroviruses, lentiviruses, and alphaviruses (e.g., Sindbis virus and Venezuelan Equine Encephalitis virus), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 1991, 2:320-330), defective herpes virus vector lacking a glyco-protein L gene (Patent Publication RD 371005 A), or other defective herpes virus vectors (International Patent Publication No. WO 94/21807, published September 29, 1994; International Patent Publication No. WO 92/05263, published April 2, 1994); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 1992, 90:626-630; see also La Salle et al., Science 1993, 259:988-990); and a defective adeno-associated virus vector (Samulski et al., J. Virol. 1987, 61:3096-3101; Samulski et al., J. Virol. 1989, 63:3822-3828; Lebkowski et al., Mol. Cell. Biol. 1988, 8:3988-3996).

Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors) and Invitrogen (Carlbad, California). In another embodiment, the vector can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 1987, 84:7413-7417; Feigner and Ringold, Science 1989, 337:387-388; Mackey et al, Proc. Natl. Acad. Sci. U.S.A. 1988, 85:8027-8031; Ulmer et al, Science 1993, 259:1745-1748). Useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127. Lipids may be chemically coupled to other molecules for the purpose of targeting (see, Mackey et al., Proc. Natl. Acad. Sci. U.S.A. 1988, 85:8027-8031). Targeted peptides, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO 96/25508), or a cationic polymer (e.g., International Patent Publication WO 95/21931).

It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art; e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 1992, 267:963-967; Wu and Wu, J. Biol. Chem. 1988, 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990; Williams et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88:2726-2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al, Hum. Gene Ther. 1992, 3:147-154; Wu and Wu, J. Biol. Chem. 1987, 262:4429-4432). U.S. Patent Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir et al., C.P. Acad. Sci. 1998, 321:893; WO 99/01157; WO 99/01158; WO 99/01175).

Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-7), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g., Wilson, Nat. Med. 1995, 1:887-889). In that regard, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

Diagnostic Methods

According to the present invention, mutated forms of PTPNl 1 can be detected to diagnose Noonan syndrome.

Accordingly, diagnostic methods may comprise, for example, detecting a mutation in a PTPNl 1 gene, wherein the mutation results in increased PTPNl 1 activity. The mutation may especially affect a coding region of the gene, such as a region of the PTPNl 1 gene that encodes a SH2 (src-homology 2) domain of the

PTPNl 1 protein, or a region of the PTPNl 1 gene that encodes a PTP (phosphotyrosine phosphatase) domain of the PTPNl 1 protein. The mutation may be a missense mutation, preferably a missense mutation resulting in nucleic acid substitution, or a deletion, or a combination of both. Preferably, the mutation results in one or more of the amino acid substitutions or deletions set forth in Table 1. Most preferably, the nucleotide substitutions or deletions are selected from the ones described in Table 1.

The diagnostic methods of the invention also encompass detecting a mutation in PTPNl 1 protein, in particular a mutation that results in increased activity of the PTPNl 1 protein. The mutation is preferably an amino acid substitution. More preferably, the mutation is in the SH2 (src-homology 2) domain of the PTPNl 1 protein, including the N-SH2 and C-SH2 domains, the domain between the N-SH2 and C-SH2 domain, or the PTP (phosphotyrosine phosphatase) domain of the PTPNl 1 protein. Preferred amino acid substitutions and deletions are set forth in Table 1. In a further embodiment, the diagnosis of Noonan syndrome in a subject comprises assessing the level of expression or activity of PTPNl 1 protein in the test subject and comparing it to the level of expression or activity in a control subject, wherein an increased expression and/or activity of the PTPNl 1 protein in the test subject compared to the control subject is indicative of Noonan syndrome. The level of expression of PTPNl 1 may be assessed by determining the amount of mRNA that encodes the PTPNl 1 protein in a biological sample, or by determining the concentration of PTPNl 1 protein in a biological sample. The level of activity of PTPNl 1 protein may be assessed by determining the level of phosphatase activity of the protein. The invention also provides kits for performing these diagnostic methods. A particular subject of the invention is a kit for diagnosing Noonan syndrome, comprising an oligonucleotide that specifically hybridizes to a site harboring a mutation of the PTPNl 1 gene, or an adjacent site, wherein the mutation results in increased basal activity of the PTPNl 1 protein. The site of mutation may particularly comprise a nucleotide selected from the group consisting of nucleotides 214, 215, 236, 844, 922, and 1510 of SEQ ED NO:l, or any nucleotide recited in Table 1, as described below. A further subject of the invention is a kit for diagnosing Noonan syndrome, comprising an antibody that specifically recognizes a mutated form of PTPNl 1 protein that results in increased basal activity of the protein. As used herein, the term "diagnosis" refers to the identification of the disease at any stage of its development, and also includes the determination of a predisposition of a subject to develop the disease. Importantly, the invention permits genetic counseling of prospective parents and in utero genetic testing for Noonan syndrome. Families with one affected parent or with advanced paternal age are of particular concern. The diagnostic method of the invention also allows confirmation of a questionable NS diagnosis based on phenotype (appearance and symptomology). The diagnostic method of the invention may also be envisioned in the case of fetal abnormalities whose cause may not be obvious, or in the case of fetal loss, to evaluate viability of future pregnancies.

The term "biological sample" refers to any cell source from which DNA may be obtained. Non-limiting examples of cell sources available in clinical practice include without limitation blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy. Cells may also be obtained from body fluids, including without limitation blood, plasma, serum, lymph, milk, cerebrospinal fluid, saliva, sweat, urine, feces, and tissue exudates (e.g., pus) at a site of infection or inflammation. For prenatal testing, genetic material can be obtained from fetal cells, e.g., from amniotic fluid (through amniocentesis), chronic villi, blood, or any tissue of a pregnant woman. DNA is extracted using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source. Generally, the minimum amount of DNA to be extracted for use in the present invention is about 25 pg (corresponding to about 5 cell equivalents of a genome size of 4 x IO⁹ base pairs). Various methods for detecting such mutated forms of PTPNl 1 are described herein.

The present invention especially contemplates detecting abnormalities, i.e., mutations in the PTPNl 1 gene that result in an increased basal activity of the PTPNl 1 protein, render the protein in a constitutively active conformation, provides prolonged increased PTPNl 1 activity, or increases the level of expressed PTPNl 1 protein.

Mutations may include an insertion in the gene, a truncation of or deletion in the gene, a nonsense mutation, a frameshift mutation, a splice-site mutation, and a missense mutation. Such mutations can occur in the coding region of the PTPNl 1 gene, more particularly in any of the functional domains, as well as in the untranslated regions, more particularly in the promoter or enhancer regions. Preferred mutations are those in any of exons 2, 3, 4, 7, 8, or 13. Even more preferred are mutations resulting in amino acid substitutions. Specific mutations are listed in Table 1:

TABLE 1

Mutations in PTPN11 Gene (n = 91). Nucleotide and amino acid substitutions are numbered as set forth in SEQ ED NO: 1 and 2, respectively.

position 179.

Nucleic Acid Based Assays

According to the invention, mutated forms of PTPNl 1 nucleic acids, i.e. in the PTPNl 1 DNA or in its transcripts, as well as a deregulated expression, e.g. overexpression, of PTPNl 1.

Standard methods for analyzing the nucleic acid contained in a biological sample and for diagnosing a genetic disorder can be employed, and many strategies for genotypic analysis are known to those of skilled in the art.

In a preferred embodiment, the determination of mutations in the PTPNl 1 gene encompasses the use of nucleic acid sequences such as specific oligonucleotides, to detect mutations in PTPNl 1 genomic DNA or mRNA in a biological sample. Such oligonucleotides may be specifically hybridize to a site of mutation, or to a region adjacent to this site of mutation present in a PTPNl 1 nucleic acid. One may also employ primers that permit amplification of all or part of PTPNl 1. Alternatively, or in combination with such techniques, oligonucleotide sequencing described herein or known to the skilled artisan can be applied to detect the PTPNl 1 mutations.

One skilled in the art may use hybridization probes in solution and in embodiments employing solid-phase procedures. In embodiments involving solid- phase procedures, the test nucleic acid is adsorbed or otherwise affixed to a selected matrix or surface. The fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes.

In another embodiment, one skilled in the art may use oligonucleotide primers in an amplification technique, such as PCR or reverse-PCR ("reverse polymerase chain reaction"), to specifically amplify the target DNA or mRNA, respectively, that is potentially present in the biological sample.

Useful oligonucleotides include primers that permit amplification of PTPNl 1 exons, such as: Exon 1:

Forward primer: 5'-GCTGACGGGAAGCAGGAAGTGG-3' (SEQ LD NO:3) Reverse primer: 5'-CTGGCACCCGTGGTTCCCTC-3'(SEQ ED NO:4)

Exon 2: Forward primer: 5'-ACTGAATCCCAGGTCTCTACCAAG -3 '(SEQ ED NO:5) Reverse primer: 5'-CAGCAAGCTATCCAAGCATGGT-3'(SEQ ID NO:6)

Exon 3:

Forward primer: 5'-CGACGTGGAAGATGAGATCTGA-3'(SEQ ID NO:7) Reverse primer: 5'-CAGTCACAAGCCTTTGGAGTCAG-3'(SEQ ID NO:8)

Exon 4:

Forward primer: 5'-GATTGATCAATCCCTTGGAGGAATG-3'(SEQ ID NO:9) Reverse primer: 5'-GTCACCAGACCCAACGTGGTG-3'(SEQ TD NO:10)

Exon 5:

Forward primer: 5'-CTGCAGTGAACATGAGAGTGCTTG-3'(SEQ ED NO: 11)

Reverse primer: 5'-GTTGAAGCTGCAATGGGTACATG-3 (SEQ ID NO:12) Exon 6:

Forward primer: 5'-TGCATTAACACCGTTTTCTGT-3'(SEQ TD NO: 13) Reverse primer: 5 '-GTCAGTTTCAAGTCTCTCAGGTC-3 '(SEQ ID NO: 14)

Exon 7: Forward primer: 5'-GAACATTTCCTAGGATGAATTCC-3'(SEQ ED NO:15) Reverse primer: 5'-GGTACAGAGGTGCTAGGAATCA-3'(SEQ TD NO:16)

Exon 8: Forward primer: 5'-GACATCAGGCAGTGTTCACGTTAC-3'(SEQ TD NO: 17) Reverse primer: 5'-CCTTAAAGTTACTTTCAGGACATG-3'(SEQ ID NO:18)

Exon 9: Forward primer: 5'-GTAAGCTTTGCTTTTCACAGTG-3'(SEQ ED NO: 19)

Reverse primer: 5'-CTAAACATGGCCAATCTGACATGTC-3'(SEQ ID NO:20)

Exon 10:

Forward primer: 5'-GCAAGACTTGAACATTTGTTTGTTGC-3'(SEQ LD NO:21) Reverse primer: 5'-GACCCTGAATTCCTACACACCATC-3'(SEQ ID NO:22)

Exon 11:

Forward primer: 5'-CAAAAGGAGACGAGTTCTGGGAAC-3'(SEQ TD NO:23) Reverse primer: 5'-GCAGTTGCTCTATGCCTCAAACAG-3'(SEQ LD NO:24)

Exon 12:

Forward primer: 5'-GCTCCAAAGAGTAGACATTGTTTC-3'(SEQ ED NO:25)

Reverse primer: 5'-GACTGTTTTCGTGAGCACTTTC-3'(SEQ ED NO:26) Exon 13:

Forward primer: 5'-CAACACTGTAGCCATTGCAACA-3'(SEQ ID NO:27) Reverse primer: 5'-CGTATCCAAGAGGCCTAGCAAG-3'(SEQ LD NO:28)

Exon 14: Forward primer: 5 '-ACCATTGTCCCTCACATGTGC-3 '(SEQ ED NO:29)

Reverse primer: 5'-CAGTGAAAGGCATGTGCTACAAAC-3'(SEQ LD NO:30)

Exon 15:

Forward primer: 5'-CAGGTCCTAGGCACAGGAACTG-3'(SEQ LD NO:31) Reverse primer: 5'-ACATTCCCAAATTGCTTGCCT-3'(SEQ ED NO:32)

The present invention is more particularly directed to a method of in vitro diagnosis of NS comprising the steps of: a) contacting a biological sample containing DNA with specific oligonucleotides permitting the amplification of all or part of the PTPNl 1 gene, the

DNA contained in the sample having being rendered accessible, where appropriate, to hybridization, and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample; b) amplifying said DNA; c) detecting the amplification products; d) comparing the amplified products as obtained to the amplified products obtained with a normal control biological sample, and thereby detecting a possible abnormality in the PTPNl 1 gene. The method of the invention can also be applied to the detection of an abnormality in the transcript of the PTPNl 1 gene, e.g. by amplifying the mRNAs contained in a biological sample, for example by RT-PCR.

Thus another subject of the present invention is a method of in vitro diagnosis of NS, as previously defined comprising the steps of : a) producing cDNA from mRNA contained in a biological sample; b) contacting said cDNA with specific oligonucleotides permitting the amplification of all or part of the transcript of the PTPNl 1 gene, under conditions permitting a hybridization of the primers with said cDNA; c) amplifying said cDNA; d) detecting the amplification products; e) comparing the amplified products as obtained to the amplified products obtained with a normal control biological sample, and thereby detecting a possible abnormality in the transcript of the PTPNl 1 gene. For RNA analysis, the biological sample may be any cell source, as described above, such as a biopsy tissue, from which RNA is isolated using standard methods well known to those of ordinary skill in the art such as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznski et al, Anal. Biochem., 1987, 162:156). The isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a selected site. Conditions for primer annealing are chosen to ensure specific reverse transcription and amplification; thus, the appearance of an amplification product is diagnostic of the presence of a particular genetic variation. In another embodiment, RNA is reverse-transcribed and amplified, after which the amplified sequences are identified by, e.g., direct sequencing. In still another embodiment, cDNA obtained from the RNA can be cloned and sequenced to identify a mutation.

The PTPNl 1 nucleic acids of the invention can also be used as probes, e.g., in therapeutic and diagnostic assays. For instance, the present invention provides a probe comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region having a nucleotide sequence that is capable of hybridizing specifically to a region of a PTPNl 1 gene which differs from that of the wild-type gene (SEQ ED NO: 33), e.g., a mutant or polymorphic region. Such probes can then be used to specifically detect which mutation of the PTPNl 1 gene is present in a sample taken from a subject. The mutant or polymorphic region can be located in the promoter, exon, or intron sequences of the PTPNl 1 gene.

For example, preferred probes of the invention include one or more of the nucleotide substitutions listed in Table 1, as well as the wild-type flanking regions (see, e.g., SEQ ED NOS: 1 or 33). For each such probe, the complement of that probe is also included in the Table as a preferred probe of the invention. Particularly preferred probes of the invention have a number of nucleotides sufficient to allow specific hybridization to the target nucleotide sequence. Thus, probes of suitable lengths based on SEQ ID NO:l or 33 and complementary to the mutant sequences provided herein can be constructed and tested by the skilled artisan for appropriate level of specificity depending on the application intended. Where the target nucleotide sequence is present in a large fragment of DNA, such as a genomic DNA fragment of several tens or hundreds of kilobases, the size of the probe may have to be longer to provide sufficiently specific hybridization, as compared to a probe which is used to detect a target sequence which is present in a shorter fragment of DNA. For example, in some diagnostic methods, a portion of the PTPNl 1 gene may first be amplified and thus isolated from the rest of the chromosomal DNA and then hybridized to a probe. In such a situation, a shorter probe will likely provide sufficient specificity of hybridization. For example, a probe having a nucleotide sequence of about 10 nucleotides may be sufficient, although probes of about 15 nucleotides, even more preferably 20 nucleotides, are preferred.

In a preferred embodiment, the probe or primer further comprises a label attached thereto, which preferably is capable of being detected. The label can, for example, be selected from radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.

In another preferred embodiment of the invention, the isolated nucleic acid, which is used, e.g., as a probe or a primer, is modified, such as to become more stable. Exemplary nucleic acid molecules which are modified include phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775).

In yet another embodiment, one may use HPLC or denaturing HPLC (DHPLC) techniques to analyze the PTPNl 1 nucleic acids. DHPLC was developed when observing that, when HPLC 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, 1995, 5:494; Underhill, et al, Proc. Natl. Acad. Sci. USA,1996, 93:193; Doris, et al., DHPLC Workshop, 1997, Stanford University). Thus, the use of DHPLC was applied to mutation detection (Underhill, et al., Genome Research, 1997, 7:996; Liu, et al., Nucleic Acid Res., 1998, 26; 1396). DHPLC can separate heteroduplexes that differ by as little as one base pair. "Matched Ion Polynucleotide Chromatography" (MEPC), or Denaturing "Matched Ion Polynucleotide Chromatography" (DMEPC) as described in U.S. Patents No. 6,287,822 or 6,024,878, are separation methods that can also be useful in connection with the present invention.

Alternatively, one can use the DGGE method (Denaturing Gradient Gel Electrophoresis), or the SSCP method (Single Strand Conformation Polymorphism) for detecting an abnormality in the PTPNl 1 gene. 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). 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. 1994, 3:801). "HOT cleavage", 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 1988, 85:4397), can also be used. Such methods are preferably followed by direct sequencing. Advantageously, the RT-PCR method may be used for detecting abnormalities in the PTPNl 1 transcript, as it allows to visualize the consequences of a splicing mutation such as exon skipping or aberrant splicing due to the activation of a cryptic site. Preferably this method is followed by direct sequencing as well.

More recently developed techniques using microarrays, preferably microarray techniques allowing for high-throughput screening, can also be advantageously implemented for detecting an abnormality in the PTPNl 1 gene or for assaying expression of the PTPNl 1 gene. 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 said selected regions of the array, against a test sample, contacted with another of said 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,CA) 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).

The solid support on which oligonucleotides are attached may be made from glass, silicon, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other materials. One method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al, Science 1995, 270:467-470. This method is especially useful for preparing microarrays of cDNA. See also DeRisi et al, Nature Genetics 1996, 14:457-460, ; Shalon et al, Genome Res. 1996, 6:639-645; and Schena et al, Proc. Natl. Acad. Sci. USA 1995, 93:10539-11286. Another method of making microarrays is by use of an inkjet printing process to bind genes or oligonucleotides directly on a solid phase, as described, e.g., in U.S. Patent No. 5,965,352.

Other methods for making microarrays, e.g., by masking (Maskos and Southern, Nuc. Acids Res. 1992, 20:1679-1684), may also be used. 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 the PTPNl 1 gene present in the tested sample , i.e., the probe hybridizes, duplexes or binds to the PTPNl 1 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 rales or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5%o mismatch. Preferably, the polynucleotides are perfectly complementary (no mismatches). 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 1996, 274:610-614). A variety of methods are available for detection and analysis of the hybridization events. Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used to label the DNA probe, detection and analysis are carried out fluorimetrically, colorimetricalfy or by autoradiography. By observing and measuring emitted radiation, such as fluorescent radiation or a particle emission, information may be obtained about the hybridization events.

When fluorescently labeled probes are used, the fluorescence emissions at each site of transcript 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. 1996, 6:639-695).

Protein Based Assays As an alternative to analyzing PTPNl 1 nucleic acids, one can evaluate

PTPNl 1 on the basis of mutations in the protein, or dysregulated production, e.g. overproduction, of the protein.

In a preferred embodiment, PTPNl 1 is detected by immunoassay. For example, Western blotting permits detection of a specific variant, or the presence or absence of PTPNl 1. In particular, an immunoassay can detect a specific (wild-type or mutant) amino acid sequence in a PTPNl 1 protein. Other immunoassay formats can also be used in place of Western blotting, as described below for the production of antibodies. One of these is ELISA assay. hi ELISA assays, an antibody against PTPNl 1 or epitopic fragment thereof is immobilized onto a selected surface, for example, a surface capable of binding proteins such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed polypeptides, a nonspecific protein such as a solution of bovine serum albumin (BSA) may be bound to the selected surface. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific bindings of antisera onto the surface. The immobilizing surface is then contacted with a sample, to be tested in a manner conductive to immune complex (antigen/antibody) formation. This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from 2 to 4 hours, at temperatures between about 25° to 37°C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or borate buffer. Following formation of specific immunocomplexes between the test sample and the bound antibody, and subsequent washing, the occurrence, and an even amount of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody against PTPNl 1, that recognizes a different epitope on the protein. To provide detecting means, the second antibody may have an associated activity such as an enzymatic activity that will generate, for example, a color development upon incubating with an appropriate chromogenic substrate. Quantification may then be achieved by measuring the degree of color generation using, for example, a visible spectra spectrophotometer.

Typically the detection antibody is conjugated to an enzyme such as peroxidase and the protein is detected by the addition of a soluble chromophore peroxidase substrate such as tetiamethylbenzidine followed by 1 M sulfuric acid. The test protein concentration is determined by comparison with standard curves.

These protocols are detailed in Current Protocols in Molecular Biology, V. 2 Ch. 11 and Antibodies, a Laboratory Manual, Ed Hariow, David Lane, Cold Spring Harbor Laboratory (1988) pp 579-593. Alternatively, a biochemical assay can be used to detect expression, or accumulation of PTPNl 1, e.g., by detecting the presence or absence of a band in samples analyzed by polyacrylamide gel electrophoresis; by the presence or absence of a chromatographic peak in samples analyzed by any of the various methods of high performance liquid chromatography, including reverse phase, ion exchange, and gel permeation; by the presence or absence of PTPNl 1 in analytical capillary electrophoresis chromatography, or any other quantitative or qualitative biochemical technique known in the art. The immunoassays discussed above involve using antibodies directed against the PTPNl 1 protein or fragments thereof. The production of such antibodies is described below.

Anti-PTPNll Antibodies

Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fab expression library.

Various procedures known in the art may be used for the production of polyclonal antibodies to PTPNl 1 polypeptides or derivative or analog thereof. For the production of antibody, various host animals can be immunized by injection with the antigenic polypeptide, including but not limited to rabbits, mice, rats, sheep, goats, etc.

For preparation of monoclonal antibodies directed toward the PTPNl 1 polypeptides, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495- 497, 1975), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today 4:72, 1983; Cote et al, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690, published 28 December, 1989).

According to the invention, techniques described for the production of single chain antibodies (U.S. Patent Nos. 5,476,786 and 5,132,405 to Huston; U.S. Patent 4,946,778) can be adapted to produce the PTPNl 1 pofypeptide-specific single chain antibodies. Indeed, these genes can be delivered for expression in vivo. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al, Science 246:1275-1281, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for a PTPNl 1 polypeptide, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragment which can be produced by pepsin t digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

PTPN11 Activity Assays In another embodiment one may assess the activity of the PTPNl 1 protein in a test subject or biological sample taken from the subject and compare it with a control. An increased activity of the PTPNl 1 protein in the test subject or biological sample compared with the control is indicative of NS in the test subject.

The activity of PTPNl 1 may be indirectly assayed by evaluating the level of expression, accumulation or activity of down-stream effectors, as described above. Preferred target are MAP kinases, such as ERK1 or ERK2. The nucleic acid- based assays or protein-based assays as described above may be readily adapted for that purpose.

Since PTPNl 1 is a phosphatase, the basal activity of PTPNl 1 in a test subject may be easily determined by assessing the phosphorylation level of peptides or proteins contacted with the test PTPNl 1 protein. For example, phosphorylation of PTPNl 1 docking partners such as Gabl, Gab2, Grb2, or gpl30, as well as peptides such as src, may be assessed. The levels of phosphorylation of proteins can be assessed by various methods, including immunoassays or radiolabelling. Kits for assessing phosphorylation activity are commercially available, e.g., from from Upstate Biotechnology, Inc. (Lake Placid, NY) under the name "PTP Assay Kit 1", and from Calbiochem (San Diego, CA) under the name "Fluorometric Protein Tyrosine Phosphatase Assay Kit." One example of a PTPase activity assay is as follows: To activate

PTPase activity, a synthesized phosphotyrosine peptide (Genemed Synthesis), PY627PY659, is used over a concentration range of 0-20 μM. PTPase reactions are carried out in 25 μl mixtures at 30°C for 5 min in 50 mM Hepes (pH 7.2), 0.2% BSA, 1 mM EDTA, and 1 mM DTT, and the reactions stopped by addition of 20 μl Malachite Green/Tween-20 solution. After 30 min at RT, absorbance at 620 nm is determined with a microplate reader. A standard curve of free phosphate concentrations is prepared using KH₂PO₄, treated identically to the PTPase conditions. All conditions are repeated in triplicate.

In one embodiment, the level of phosphorylation of a peptide or protein is assessed by utilizing a binding partner, which should preferably be highly specific for the phosphoepitope on the target protein. It is preferred that the binding partner is an antibody. The antibody is preferably generated against a unique epitope of the substrate. In an alternative embodiment, the binding partner should he specific for the phosphorylated form of the target protein. The detection procedure used to assess the phosphorylation state of the protein may for instance employ an antibody or a peptide that recognizes and binds to phosphorylated serines, threonines or tyrosines. The detection antibody is preferably a polyclonal antibody, to maximize the signal, but may also be specific monoclonal antibodies which have been optimized for signal generation. An exemplary PTPase assay based on immunoprecipitation is as follows:

Step 1: Immunoprecipation of Phosphatase

To maximize Phosphatase activity, all reactions should be carried out on ice, and pulse spinning should be carried out in a centrifuge equilibrated at 4°C. 1. Add 4μl-5μl of an anti-Phosphatase antibody to a microcentrifuge tube. 2. Add lOOμl (50μl packed beads) of Protein A or G agarose bead slurry, (which has been washed free of phosphate) and suspended in a cell lysis buffer optimized for phosphatases.

3. Add 100 μl of ice-cold phosphatase cell lysis buffer (optionally containing protease inhibitors, but must be free of phosphatase inhibitors).

4. Incubate for 30 minutes to 1 hour at 4°C on a rotator to thoroughly mix the components during the incubation.

5. Pellet the agarose beads at 14,000 rpm for 15 seconds.

6. Remove the supernanant. Wash the protein A or G agarose beads in cell lysis buffer to remove weakly bound antibodies. This wash removes any form of antibody that does not bind to the protein A or G agarose, but which may bind to a tissue extract or cell lysate.

7. Resuspend the washed beads pellet in lOOμl of phosphatase cell lysis buffer. 8. Add whole cell/tissue extracts (about 500μg-lmg) containing active phosphatase to the beads.

9. Incubate for 2 hours on a rotator at 4°C to immunoprecipitate active phosphatase.

10. Wash the protein A or G agarose/enzyme immunocomplex two to three times with 500/xl of phosphatase cell lysis buffer.

11. Wash the protein A or G agarose/enzyme immunocomplex twice with lOOμl of lx phosphatase assay dilution buffer found to be suitable (e.g., HEPES, glycerol and EDTA buffer can be used as a start, and modified as necessary).

12. Pellet the immunocomplex at 14,000 rpm for 15 minutes and remove the supernatant fraction from the immunocomplex. Place on ice and proceed to Step II.

Step II: Malachite Green Phosphatase Assay of the Enzyme Immunocomplex

1. Add 50μl of a lx phosphatase buffer containing phosphothreonine peptide (KRμTIRR) to the immunocomplex.

2. Incubate overnight at 37°C or, if preferred, for 30 minutes in a 30°C shaking incubator. After the incubation, pulse spin to pellet the protein A or G - Agarose/enzyme immunocomplex. Note: the assay mixture must be thoroughly mixed throughout the reaction time to ensure that the peptide substrate and the enzyme immunocomplex achieve maximum interaction.

3. Add a suitable amount (e.g., 1, 2, 5, or 10 μl) of the supernatant into a 96 well plate and add Malachite Green solution (Van Veldhoven et al., Anal Biochem 1987;161:45-48) to assess for the liberation of phosphate measured in picomoles from the standard curve.

To assess for specific phosphatase activity, the phosphate levels detected with protein A or G-agarose beads incubated with cell lysates in the absence of antibody can be used as a control. Any buffer components should be checked for free phosphate, and the assay optimized as to not inhibit phosphatase activity. In addition, because the assay measures free phosphate, phosphate buffers are not compatible with this system. Thus, reaction components that contain phosphate (i.e., glycerol phosphate) may interfere with the analysis, depending on their concentration, purity and stability in strong acid. Also, high concentrations of reductants may bleach the dye color over time resulting in lower sensitivity. A final concentration of 0.02%> p-mercaptoethanol has no effect on sensitivity; 0.05%> p-mercaptoethanol has only a slight effect, and 0.1 %> p-mercaptoethanol results in an approximate 20% reduction in sensitivity. Many detergents at or below 0.1% can be used, but higher concentrations may generate high backgrounds. If high concentrations of detergent are required in the reaction, the background can be determined by including the corresponding concentration of the detergent in the Phosphate Standard curve.

Alternatively, immunoassays may be replaced by the detection of radiolabeled phosphate according to a standard technique. This involves incubating cells with the test substances and radiolabeled phosphate, lysing the cells, separating cellular protein components of the lysate using as SDS-polyacrylamide gel (SDS- PAGE) technique, in either one or two dimensions, and detecting the presence of phosphorylated proteins by exposing X-ray film.

The phosphorylation of a protein may also be conveniently detected by migration on gel subject to electrophoresis, followed by Western blotting. Phosphorylation is detected by a shift of the molecular weight of the protein occurs, a phosphorylated protein being heavier than the corresponding non-phosphorylated form. Diagnostic Kits

The present invention further provides kits for the determination of the sequence within the PTPNl 1 gene in an individual. The kits comprise a means for determining the sequence at the variant positions, and may optionally include data for analysis of mutations. The means for sequence determination may comprise suitable nucleic acid-based and immunological reagents. Preferably, the kits also comprise suitable buffers, control reagents where appropriate, and directions for determining the sequence at a variant position.

Nucleic Acid Based Diagnostic Kits

The invention provides nucleic acid-based methods for detecting genetic variations of PTPNl 1 in a biological sample. The sequence at particular positions in the PTPNl 1 gene is determined using any suitable means known in the art, including without limitation one or more of hybridization with specific probes for PCR amplification (e.g., primer pairs selected from SEQ ED NOS:3-32), restriction fragmentation, direct sequencing, SSCP, and other techniques known in the art.

The present invention also provides kits suitable for nucleic acid-based diagnostic applications. In one embodiment, diagnostic kits include the following components: a) Probe DNA: The probe DNA may be pre-labeled; alternatively, the probe DNA may be unlabeled and the ingredients for labeling may be included in the kit in separate containers; and b) Hybridization reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.

In another embodiment, diagnostic kits include: a) Sequence determination primers: Sequencing primers may be pre- labeled or may contain an affinity purification or attachment moiety; and b) Sequence determination reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular sequencing protocol.

In one preferred embodiment, the kit comprises a panel of sequencing primers, whose sequences correspond to sequences adjacent to variant positions. Antibody Based Diagnostic Kits

The invention also provides antibody-based methods for detecting mutant (or wild type) PTPNl 1 proteins in a biological sample. The methods comprise the steps of: (i) contacting a sample with one or more antibody preparations, wherein each of the antibody preparations is specific for mutant (or wild type) PTPNl 1 under conditions in which a stable antigen-antibody complex can form between the antibody and PTPNl 1 in the sample; and (ii) detecting any antigen-antibody complex formed in step (i) using any suitable means known in the art, wherein the detection of a complex indicates the presence of mutant (or wild type) PTPNl 1. Typically, immunoassays use either a labeled antibody or a labeled antigenic component (e.g. , that competes with the antigen in the sample for binding to the antibody). Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labeled immunoassays, such as ELISA assays.

The present invention also provides kits suitable for antibody-based diagnostic applications. Diagnostic kits typically include one or more of the following components:

(i) PTPNl 1 -specific antibodies: The antibodies may be pre- labeled; alternatively, the antibody may be unlabeled and the ingredients for labeling may be included in the kit in separate containers, or a secondary, labeled antibody is provided; and

(ii) Reaction components: The kit may also contain other suitably packaged reagents and materials needed for the particular immunoassay protocol, including solid-phase matrices, if applicable, and standards.

The kits referred to above may include instructions for conducting the test. Furthermore, in preferred embodiments, the diagnostic kits are adaptable to high-throughput and/or automated operation.

Therapeutics

The present invention further provides a method for the treatment of NS, which method comprises modulating PTPNl 1 activity in a subject or patient. The method comprises administering to a patient in need of such treatment an effective amount of an agent that modulates PTPNl 1 expression or activity, with a pharmaceutically acceptable carrier. For example, the therapeutic agent may be a PTPNl 1 antisense nucleic acid, or an anti-PTPNl intracellular inhibitory antibody. Agents that block either the N-SH2, C-SH2, or PTP domains of the PTPNl 1 proteins are of particular interest. Preferably, although not necessarily, the agent blocks the PTP domain so that PTPase activity is inhibited.

A "subject" or "patient" is a human or an animal likely to develop NS, more particularly a mammal, preferably a rodent or a primate, as described above in connection with diagnostic applications. Prenatal treatment is particularly envisioned. The term "treatment" means to therapeutically intervene in the development of a disease in a subject showing a symptom of this disease. The term "treatment" also encompasses prevention, which means to prophylactically interfere with a pathological mechanism that results in the disease.

The term "modulating PTPNl 1 activity" in a subject means modifying it so that it is rendered as close as possible to the normal PTPNl 1 activity of a control subject. It especially encompasses inhibiting, or blocking the activity of the PTPNl 1 protein in the NS patient. Preferred modulators block any of the functional domains of the PTPNl 1 of the protein, especially the consitutively active PTP domain. "Modulating PTPNl 1 activity" also encompasses restoring SH2 domain activity. The modulation activity may be achieved by various methods, as described hereafter.

In one embodiment, the modulatory agent may be a substance that is known or has been identified to modulate, especially inhibit, whether fully or partially, PTPNl 1 activity. Such compounds can include any compound(s) described in, for example, the International Patent Publication WO99/46267 and in Ann Rev Pharmocol Toxicol 2002;42:209-234; Exp Mol Med 2002;31:211-223; Biochem 2002;41:10700-10709; and J Immunol 167:3391-3397, 2001, as well as other compounds shown to inhibit PTPNl activity as described herein. For example, this modulatory agent may be a candidate drug as identified by a screening method. It may also be an inhibitory antibody directed against PTPNl 1. In a further embodiment, it may be an antisense nucleic acid. All these embodiments are described in greater detail below.

The term "therapeutically effective amount" is used herein to mean an amount or dose sufficient to modulate, e.g., decrease the level of PTPNl 1 activity e.g., by about 10 percent, preferably by about 50 percent, and more preferably by about 90 percent. Preferably, a therapeutically effective amount can ameliorate or present a clinically significant deficit in the activity, function and response of the subject. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.

The substance that modulates or inhibits PTPNl 1 activity is advantageously formulated in a pharmaceutical composition, with a pharmaceutically acceptable carrier. This substance may be then called active ingredient or therapeutic agent against NS. The concentration or amount of the active ingredient depends on the desired dosage and administration regimen, as discussed below. Suitable dose ranges may include from about 0.01 mg/kg to about 100 mg/kg of body weight per day.

The pharmaceutical compositions may also include other biologically active compounds. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

A composition comprising "A" (where "A" is a single protein, DNA molecule, vector, recombinant host cell, etc.) is substantially free of "B" (where "B" comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A". Preferably, "A" comprises at least about 90%) by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.

According to the invention, the pharmaceutical composition of the invention can be introduced parenterally, transmucosally, e.g., orally (per os), nasally, or rectally, or transdermally. Parental routes include intravenous, intra-arteriole, intra-muscular, intradermal, subcutaneous, intraperitoneal, mtraventricular, and intracranial administration. Targeting heart, e.g. by direct administration to heart muscle or cavities, may be advantageous. The pharmaceutical compositions may be added to a retained physiological fluid such as blood or synovial fluid.

In another embodiment, the active ingredient can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.

317-327; see generally ibid.).

In yet another embodiment, the therapeutic compound can be delivered in a controlled release system. For example, a polypeptide may be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly- lactic/glutamic acid (PLGA), a pellet containing a mixture of cholesterol and the active ingredient (SilasticR™; Dow Corning, Midland, MI; see U.S. Patent No.

5,554,601) implanted subcutaneously, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.

Screening Methods

A "test substance" is a chemically defined compound or mixture of compounds (as in the case of a natural extract or tissue culture supernatant), whose ability to modulate PTPNl 1 activity may be defined by various assays. A "test substance" is also referred to as a "candidate drug" in the present description. Test substances may be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, Wl). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TEBTech 1996, 14:60).

A modulatory effect may be determined by an in vitro method using a recombinant PTPNl 1 -reporter gene promoter activity system. Reporter genes for use in the invention encode detectable proteins, include, but are by no means limited to, chloramphenicol transferase (CAT), β- galactosidase (/3-gaI), luciferase, green fluorescent protein (GFP) and derivatives thereof, yellow fluorescent protein and derivatives thereof, alkaline phosphatase, other enzymes that can be adapted to produce a detectable product, and other gene products that can be detected, e.g., immunologically (by immunoassay).

A screen according to the invention involves detecting expression of the reporter gene by the host cell when contacted with a test substance. If there is no change in expression of the reporter gene, the test substance is not an effective modulator. If reporter gene expression is modified, in particular reduced or eliminated, the test substance has modulated, e.g., inhibited, PTPNl 1 -mediated gene expression, and is thus a candidate for development of an NS therapeutic.

The reporter gene assay system described here may be used in a high- throughput primary screen for antagonists, or it may be used as a secondary functional screen for candidate compounds identified by a different primary screen, e.g., a binding assay screen that identifies compounds that modulate PTPNl 1 transcription activity.

Potential drugs may be identified by screening in high-throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time (see, e.g., U.S. Patent Nos. 5,585,277, 5,679,582, and 6,020,141). Such high-throughput screening methods are particularly preferred. Alternatively, simple reporter-gene based cell assays such as the one described here are also highly desirable.

Intact cells or whole animals expressing a gene encoding PTPNl 1 can be used in screening methods to identify candidate drugs. In one series of embodiments, a permanent cell line is established.

Alternatively, cells are transiently programmed to express a PTPNl 1 gene by introduction of appropriate DNA or mRNA.

Identification of candidate substances can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to PTPNl 1 (ii) assays that measure the ability of a test substance to modify (i.e., inhibit) a measurable activity or function of PTPNl 1 and (iii) assays that measure the ability of a substance to modify (i.e., inhibit) the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions of the PTPNl 1 gene.

Selected agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways, e.g. to enhance their proteolytic stability.

Inhibitory Antibodies

The modulatory substance may also be an antibody that is directed against PTPNl 1. Antibodies that block the activity of PTPNl 1 may be produced and selected according to any standard method well-known by one skilled in the art, such as those described above in the context of diagnostic applications. Intracellular antibodies (sometime referred to as "intrabodies") have been used to regulate the activity of intracellular proteins in a number of systems (see, Marasco, Gene Ther. 1997, 4:11; Chen et al., Hum. Gene Ther. 1994, 5:595), e.g., viral infections (Marasco et al., Hum. Gene Ther. 1998, 9:1627) and other infectious diseases (Rondon et al., Annu. Rev. Microbiol. 1997, 51:257), and oncogenes, such as p21 (Cardinale et al., FEBS Lett. 1998, 439:197-202; Cochet et al., Cancer Res. 1998, 58:1170-6), myb (Kasono et al., Biochem Biophys Res Commun. 1998, 251:124-30), erbB-2 (Graus-Porta et al., Mol Cell Biol. 1995, 15:1182-91), etc. This technology can be adapted to inhibit PTPNl 1 activity by expression of an anti-PTPNll intracellular antibody. Antisense Therapy

Ln another embodiment, vectors comprising a sequence encoding an antisense nucleic acid according to the invention may be administered by any known methods, such as the methods for gene therapy available in the art. Exemplary methods are described below. For general reviews of the methods of gene therapy, see, Goldspiel et al, Clinical Pharmacy 1993, 12:488-505; Wu and Wu, Biotherapy 1991, 3:87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 1993, 32:573-596; Mulligan, Science 1993, 260:926-932; and Morgan and Anderson, Ann. Rev. Biochem. 1993, 62:191-217; May, TIBTECH 1993, 11:155-215. Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al, (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al, (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.

In one embodiment, a vector is used in which the coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for expression of the construct from a nucleic acid molecule that has integrated into the genome (Koller and Smithies, Proc. Natl. Acad. Sci. USA 1989, 86:8932-8935; Zijlstra et al, Nature 1989, 342:435-438).

Delivery of the vector into a patient may be either direct, in which case - the patient is directly exposed to the vector or a delivery complex, or indirect, in which case, cells are first transformed with the vector in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo and ex vivo gene therapy.

In a specific embodiment, the vector is directly administered in vivo, where it enters the cells of the organism and mediates expression of the construct. This can be accomplished by any of numerous methods known in the art and discussed above, e.g., by constructing it as part of an appropriate expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in biopolymers (e.g., poly-β-l-→4-N- acetylglucosamine polysaccharide; see , U.S. Patent No. 5,635,493), encapsulation in liposomes, microparticles, or microcapsules; by administering it in linkage to a peptide or other ligand known to enter the nucleus; or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 1987, 62:4429-4432), etc. hi another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation, or cationic 12-mer peptides, e.g., derived from antem apedia, that can be used to transfer therapeutic DNA into cells (Mi et al, Mol. Therapy 2000, 2:339-47). In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publication Nos. WO 92/06180, WO 92/22635, WO 92/20316 and WO 93/14188).

Preferred examples of specific ∑ωti-PTPNll antisense sequences are any of the sequences described in U.S. Patent No. 6,200,807, owned by Isis Pharmaceuticals. Antisense compounds that are 8 to 30 nucleobases in length and are targeted to a region selected from the 5' untranslated region, the start codon, the region from nucleotides 298 through 1883 of the coding region, the stop codon, or the 3' untranslated region of human PTPNl 1, are of particular interest. Examples of practicing the invention are provided, and are understood to be exemplary only, and do not limit the scope of the invention or the appended claims. A person of ordinary skill in the art will appreciate that the invention can be practiced in many forms according to the claims and disclosures here.

EXAMPLE 1: Detection of Mutations in PTPNl 1 Gene

Methods Subject recruitment. Subjects with NS were recruited, primarily through a GeneTests listing, and informed consent was obtained from all subjects. The diagnosis of NS was assigned based on clinical evaluations from the referring clinical geneticists. Molecular analysis. Genomic DNA was isolated from peripheral blood lymphocytes (Gentra). Genotyping was carried out using dye-labeled simple tandem repeat markers, D12S84, D12S105, D12S354 and D12S2070 (Research Genetics). PCR products were resolved by using the ABI Prism 377 DNA Sequencer (Perkin Elmer), and genotype determinations were conducted using the GeneScan v. 3.1 and Genotyper v. 2.1 software packages (Perkin Elmer).

Genomic and cDNA sequences for PTPNl 1 were compared using DNA analysis software (MacVector) and predictions of the gene organization were made using the NIX software package (UK HGMRP). Mutational screening of PTPNl 1 was carried out by direct sequencing of purified PCR products bi- directionally using the ABI BigDye terminator Sequencing Kit (Perkin Elmer) and the ABI 3700 Capillary Array Sequencer (Perkin Elmer). Sequences were analyzed using Sequencing Analysis v. 3.6.1 and AutoAssembler v.1.4.0 software packages (Perkin Elmer). To confirm sequence changes in affected individuals and exclude them from unaffected family members and controls, the relevant exons were PCR amplified and then digested with restriction endonucleases (BgH for G214T and C215G, BsaWl for A236G and EcoRV for A922G) according to manufacturer's instructions (New England Biolabs), or analyzed by DHPLC (A844G and A1510G) using the Wave DNA Fragment Analysis System (Transgenomics) at column temperatures recommended by the WaveMaker version 4.0.28 software (Transgenomics).

Results

Mutation screening was initially conducted with two moderate-sized families (called NS-C and NS-L), in which the NS phenotype co-segregated with haplotypes defined by D12S84, D12S105, D12S354 and D12S2070. Bi-directional sequencing of the fifteen PTPNl 1 exons and their intron boundaries for family NS-C revealed a G-to-T transversion at position 214 in exon 3, predicting the substitution of Ala72 by a Ser residue (A72S) in the N-SH2 domain.

This sequence change was confirmed with a PCR-based RFLP assay that documented its presence in all affected family members and its absence among unaffected ones. This change was not observed in more than 200 control individuals. Sequence comparison of PTPNl 1 with its orthologues and other closely related PTPases revealed complete conservation of Ala⁷². Analysis of family NS-L revealed an A-to-G transition at position 236 in exon 3. This change predicted the substitution of Gln79 by an Arg (Q79R), affecting another highly conserved residue in the N-SH2 domain. This sequence change was confirmed in all affected individuals in this family, but was absent in unaffected family members and controls.

Sequence analysis of amplified DNA containing the PTPNl 1 exon 3 and flanking intronic boundaries that revealed heterozygous changes in the NS-C and NS-L families is shown in Figures 3A, 3B, 4A and 4B.

Since NS is genetically heterogeneous, the relative importance of

PTPNl 1 defects in the epidemiology of NS was considered. Mutation screening was performed with 14 unrelated individuals affected with NS, either sporadic cases or small kindreds for which linkage analysis would not have been informative. Among these 14, PTPNl 1 missense mutations were identified in seven (Table 2).

TABLE 2 PTPN11 and PTPN11 Mutations Among Familial And Sporadic Noonan Syndrome Cases

Family ID Nucleotide Exon Amino Functiona substitutio acid 1 domain n substitutio n

NS-C G214T 3 A72S N-SH2

NS-U C215G 3 A72G N-SH2

NS-L, NS-D A236G 3 Q79R N-SH2

NS-F A844G 7 I282V PTP

NS-A. NS-B. NS-V A922G 8 Ν308D PTP

NS-N A1510G 13 M504V PTP

Ala was altered by a second mutation, which was observed in family

ΝS-U, a C-to-G transversion at nucleotide 215 that predicted its substitution with a Gly (A72G). The Q79R mutation was recurrent in sporadic case ΝS-D. Three missense mutations affecting the PTP domain were observed, one being recurrent. An A-to-G transition at nucleotide 922 in exon 8 was found in families NS-A and NS-V as well as sporadic case NS-B. This change predicted the substitution of Asn³⁰⁸ with an Asp residue (N308D). Sporadic cases NS-F and NS-N had A-to-G transitions of nucleotides 844 and 1510, respectively, leading to substitutions of He²⁸² and Met⁵⁰⁴ with Val residues (I282V and M504V, respectively). For familial cases, mutations co- segregated with the NS phenotype, and no mutation was found among greater than 200 controls. All amino acid substitutions affected highly conserved residues of PTPNl 1. The results of this small series, combined with those from the two chromosome 12-linked families, suggest that greater than 50%> of NS cases can be attributed to PTPNl 1 mutations.

EXAMPLE 2: Molecular Consequences Of PTPNl 1 Mutations

To explore the molecular consequences of the amino acid substitutions, crystallographic information about PTPNl 1 was exploited (Lee, et al., Structure, 1994, 2:423-438; Eck et al, Nature, 1996, 379:277-280; Hof et al, Cell, 1998, 92:441-450).

Methods Computational methods. Recently, a protocol was devised for calculating the (unknown) structure of segments connecting known elements of secondary structure in proteins. The approach consists of two steps where first Monte Carlo-simulated annealing is carried out to allow the segment to find all structures that are intrinsic to its sequence, while it remains tethered to the protein only at its amino terminus. In the second step, MC-SCV (Noguti et al Biopolymers, 1985, 24:527-546) is used to drive the carboxy terminus to its attachment point. The entire procedure has been programmed into the CHARMM suite of programs (Brooks, et al. J. Comput. Chem, 1983, 4:165-175) using the PAR22 all-atom force field (MacKerell, et al. J. Phys. Chem. B, 1998, 102:3586-3616). The method is fully described in Hassan, S.A., Mehler, E.L. & Weinstein, H. Structure calculations of protein segments connecting domains with defined secondary structure: A simulated annealing Monte Carlo combined with biased scaled collective variables technique. In Lecture Notes Series in Computational Science and Engineering, Vol. 24 (eds. T. Schlick and H.H. Gan), pp. 197-231 (Springer Verlag, New York, 2002). In the present calculations, the first step of the general procedure was modified. Starting from the native conformation, the segment was cut between E76 and L77, and a long MC-SCV simulation was carried out on the nine residue segment Gly⁶⁸-Glu⁷⁶ at T = 31 OK. In this step, only a few fixed residues at the amino and carboxy termini of the variable segment were included in the calculation. This step allowed the segment to relax and explore conformation space to find conformations intrinsic to its sequence and resulted in a large number of conformations that provide the starting structures for the second step. To reduce the calculation, the structures were first clustered (Shenkin, et al. J. Comput. Chem., 1994, 15:899-916).on the basis of structural similarity and a representative from each class (n=31) was used.

In the second step, MC-SCV (Noguti et al., Biopolymers, 1985, 24:527-546) was used to drive the segment back to its attachment point at Leu⁷⁷. This was achieved by attaching a dummy residue at the carboxy terminus identical with the target residue (Leu⁷⁷) to which the segment will attach. This dummy residue had no role in the energetics but served as a geometric reference for adding a harmonic term, ∑k(rj - rj°) , to the energy that drove the segment to its target; η and rj° are the coordinates of atom i in the dummy and target residues, respectively, and the sum runs over the backbone and Cβ atoms. Starting with k=0, an MC-SCV simulation was carried out at 31 OK on each coordinate set obtained from the first step to relax it in the field of the tertiary structure of the protein that had now been added to the system, as well as the aqueous solvent represented by the screened Coulomb potential-implicit solvent model (Hassan, et al, J. Phys. Chem. B, 2000, 104:6478- 6489). Subsequently, k was increased in successive steps, with an MC-SCV simulation carried out at each step, to facilitate the movement of the carboxy terminus to its attachment point. A power schedule of l = 10kj_ι was used, starting from k₀ = 10^" . Ten steps have been found sufficient to achieve closure.

Results

When the six residues affected by NS-causing mutations were identified in the three-dimensional structure of PTPNl 1 in the inactive (I) conformation, it became clear that all resided in and around the interactive surfaces of the N-SH2 and PTP domains. Most strikingly, Ala⁷², which was affected by two mutations, resided in the loop between βF and αB and interacted directly with PTP in the I conformation by forming a hydrogen bond with the PTP domain residue, Gin⁵⁰ . These observations suggested that the A72S and A72G mutations might affect the intrinsic structural properties of the loop segment around Ala⁷², shifting the equilibrium between the A (active) and I (inactive) states of PTPNl 1. To determine the energetically accessible conformations of the segment Gly⁶⁸-Glu⁷⁶ in the context of N-SH2 in the A conformation, the technique of Monte Carlo (Allen, M.P. & Tildesley. Computer simulation of liquids (Clarendon Press, Oxford, 1987) with scaled collective variables (MC-SCV) (Noguti, T. & Go, N. Biopolymers, 1985, 24:527-546) was used. From this procedure, more than thirty structures were analyzed to relate their structure and energy. For the wild type, only three low-energy structures had Cα-root mean square distance (rmsd) <2A. The lowest energy structure (rmsd = 0.86A) was ~8 Kcal/mole below the other two states with small rmsd, supporting the observed stability of the active conformation of isolated N-SH2 (Lee, et al. Structure, 1994, 2:423-438). In addition to the two low- rmsd states around 8 Kcal/mole above the lowest energy conformation, several conformations were found at this energy with considerably larger rmsd values. Therefore, shuttling of wild-type PTPNl 1 between the A and I conformations becomes energetically plausible if, upon interacting with the PTP domain, the energy gap between the lowest energy conformation and the nearby states decreases. This would make those conformations accessible and confer the requisite flexibility to this segment of N-SH2. hi contrast, both A72G and A72S had large populations with small rmsd and low energies. Even if the energies of these states shifted relative to each other upon binding of the PTP domain, there would still be several low-energy conformations available that were close to the A conformation, but none that would confer the flexibility required for transition to the I state. Consequently, the ability of these two mutants to shuttle between the A and I states is impaired, suggesting that the equilibrium is shifted toward the A state relative to the wild-type protein.

There are functional data supporting the conclusions of the foregoing energetics-based structural analyses. O'Reilly and co-workers (O'Reilly et al, Mol. Cell. Biol. 2000, 20, 299-311) created and characterized two PTPNl 1 mutants, D61 A and E76A, which they postulated would be gain-of-function changes since both are N-SH2 residues that interact directly with the PTP domain. Both mutants showed increased basal phosphatase activity (E76A»D61A>WT) and retained normal phosphopeptide binding properties. When expressed in Xenopus ectodermal explants, both mutants induced changes mimicking some aspects of development that are fibroblast growth factor-inducible, documenting basal stimulation of some signaling cascades in vivo. D61G, found in case N-17, was extremely similar to D61A, providing strong evidence that this mutation has gain-of-function effects. E76D, observed in case N-34, affected the same residue as the E76A, but was more a more conservative change. Glu , however, is invariant among the PTPNl 1 orthologues and homologues. The similarity in function of Asp , Ala and Glu in stabilizing the I state (Hof et al, Cell, 1998, 92, 441-450) .provides further evidence that NS is caused by increased activity of PTPNl 1.

Example 3: Characterization of mutant PTPNl 1 proteins

A. Analysis Of Basal And Signal-Dependent Phosphatase Activity Of Mutated PTPNl 1 In Vitro

The full length PTPNl 1 cDNA was cloned into pRc/CMV vector (Invitrogen) and the cDNA was shuttled into pcDNA6/N5-his A vector (Introgen). Three separate mutations were introduced into the PTPΝ11-N5 fusion construct using site-directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Stratagene): A72S (A⁷² interacts directly with the PTP domain), I282N (I²⁸² interacts directly with the Ν-SH2 domain), and N308D (recurrent mutation). After sequence confirmation, the wild type and mutant PTPNl 1-V5 constructs were transfected into COS-7 cells using LipofectaminePlus (Invitrogen) according to the manufacturer's protocol. After growing the transfected cells in complete growth medium (DMEM supplemented with 10% fetal bovine serum (FBS)) for 24 h, they were starved for 20 h in DMEM with 0.1%FBS and then exposed to epidermal growth factor (EGF). Cells were lysed, and the fusion protein was purified by immunoprecipitation using anti-V5 (Invitrogen).

PTPase assays were determined by measuring the phosphate released from phosphopeptides using the Malachite Green assay (Harder et al, Biochem J, 1994;298:395-401). SrcPY 80 nM (Calbiochem), a phosphopeptide derived from the c-Src carboxyl-terminal region, was used as substrate.

After 30 min at RT, absorbance at 620 nm was determined with a microplate reader. A standard curve of free phosphate concentrations is prepared using KH₂PO₄, treated identically to the PTPase conditions. All conditions were repeated in triplicate.

As shown in Figure 7, the three mutant PTPNl 1 protein have significantly increased phosphatase activity, even without stimulation of the COS-7 cells with EGF. These results document that the PTPNl 1 mutations observed in NS result in a gain of function.

B. Analysis Of The Docking Function Of Mutated PTPNl 1

To evaluate the docking function of PTPNl 1, COS-7 cells were transfected with wild type and mutant PTPNl 1-V5 constructs as well as a FLAG- Gabl construct. After starvation for 20 h, the cells were stimulated with EGF 0.25 ng/ml for 5 min and then lysed. Anti-FLAG monoclonal antibody (Sigma) was used for immunoprecipitation. Precipitated proteins were resolved by SDS-PAGE and transferred to nitrocellulose filters. Standard immunoblotting was carried out using anti-Gab 1 (Upstate Biotechnology), anti-phosphotyrosine (Transduction Laboratories) and anti-PTPNll (Santa Cruz) antibodies. Gabl immunoblotting determined equivalence of Gabl levels. Anti-phosphotyrosine immunoblotting documents that EGF stimulation results in Gabl phosphorylation.

As shown in Figure 8 A, anti-PTPNll immunoblotting revealed that there was an increase in docking between Gabl and mutant PTPNl 1 under basal and activated conditions compared to the wild type PTPNl 1. Briefly, COS-7 cells were transfected with constructs for FLAG-Gabl and PTPNl 1-V5 (also referred to as SHP-2-V5). Gabl was immunoprecipitated with anti-FLAG antibodies from cell lysates at 0, 15, 30, and 60 min after EGF stimulation. Proteins were separated with SDS-PAGE and immunoblotted. As shown in the bottom panel of Figure 8A, the level of Gabl was nearly equal for all conditions. Comparison of the quantities of PTPNl 1 detected revealed that there was almost none docked to Gabl prior to EGF stimulation and modest amounts thereafter. By contrast, the A72S PTPNl 1 mutant co-isolated with Gabl without EGF stimulation and was present in much larger amounts compared to wild type PTPNl 1 after EGF stimulation.

To correlate and expand those findings, COS-7 cells were transfected with the PTPNl 1-V5 fusion constructs for wild type or N308D (referred to as D308) PTPNl 1-V5. Lysates from starved or EGF-stimulated cells were obtained, and PTPNl 1-V5 proteins were immunoprecipitated with anti-V5 antibody at 0, 5, 15, 30, and 60 min after EGF stimulation. Proteins were separated with SDS-PAGE, and immuno-blotting performed with specific antibodies to measure PTPNl 1 levels as well as co-precipitation of Gabl. As shown in the bottom panel of Figure 8B, roughly equivalent amount of the PTPNl 1 (referred to as V5-SH2) protein was obtained in all conditions. Figure 8B further shows that there was increased docking of the mutant PTPNl 1-V5 with Gabl under basal and activated conditions. These results provided confirmation that the NS mutations result in a gain of PTPNl l's docking function.

C. Determination Of The Effects Of Mutated PTPNl 1 On The ERK MAP Kinase Cascade

Modulation of the extracellular signal-related kinase group (ERKl and ERK2) of MAP kinases is a major feature of PTPNl 1 function for several intracellular transduction pathways (Bennett et al., Mol Cell Biol, 1996;16:1189-1202; Craddock et al., J Biol Chem, 2001;276:24274-24283; Cunnick et al., J Biol Chem 2000;275:13842-13848; Schaeper et al, J Cell Biol, 2000;149:1419-1432; and Xu et al., J Biol Chem, 2001;276:13049-13056).

To investigate the effects of NS PTPNl 1 mutations on this pathway, COS7 cells were transfected with a construct with hemagglutin-tagged ERK2 (HA- ERK2) as well as mutant or wild type PTPNl 1. Lysates were made from serum-starved and EGF-treat cells, and protein was immunoprecipitated from cell lysates from 0, 15, 30, and 60 min after EGF stimulation. PTPNl 1 levels were assessed by immunoblotting with aliquots of the lysates. Immunoprecipitation with anti-HA (Covance) was performed using the lysates. A portion of the immunoprecipitates was immunoblotted with anti-ERK2 (Jerry Wu). Another aliquot was used to assay ERK2 kinase activity by incubating at 30°C for 5-10 min in 20 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 10 mM ρ-nitroρhenyl phosphate, 40 μM ATP, 0.375 mg/ml myelin basic protein (Sigma), with 10 μCi of [α-³²P]ATP. After terminating the reactions with SDS loading buffer and heating to 95°C for 5 min, proteins were separated with SDS- PAGE. Gels were dried and autoradiography was performed.

As shown in Figure 9, the mutant PTPNl 1 proteins resulted in a prolonged increase in the kinase activity of ERK2 compared to wild type PTPNl 1. This result is consistent with increased signaling flux through the RAS-MAP kinase pathway. Since it has been shown that PTPNl 1 is a positive regulator of the EGF signal transduction pathway (Cunnick et al., J Biol Chem, 275:13842-13848, 2000), these results document another aspect of mutant PTPNl 1 gain-of-function.

EXAMPLE 4: PTPN11 Mutations in Noonans Syndrome: Molecular Spectrum, Genotype-Phenotype Correlation, and PhenotypicHeterogeneity

This Example reports the screening for PTPNl 1 coding regions in a large, well-characterized cohort of individuals with sporadic or familial NS. The results provide a more extensive assessment of the range of PTPNl 1 lesions causing NS, evaluation of disease penetrance, establishment of genotype-phenotype correlation, and broadening of the phenotype associated with PTPNl 1 mutations to include NS with multiple giant-cell lesions.

Material and Methods

Clinical Evaluation. Subjects were examined by clinicians experienced with NS. Electrocardiograms, echocardiograms, and clinical photographs were obtained routinely for the probands, as well as for most of other affected family members in the kindreds segregating the disorder. NS was diagnosed on the basis of the presence of the following major characteristics: typical facial dysmorphia, pulmonic stenosis or HCM plus abnormal electrocardiogram pattern, pectus carinatum/excavatum, height > 2 SD below the mean, and cryptorchidism in male subjects. To have a diagnosis of NS, individuals with typical facial dysmorphia had to have at least one additional major feature, whereas individuals with suggestive facial findings had to have at least two other major characteristics (van der Burgt et al. Am J Med Genet 1994;53:187-191). HCM was diagnosed when the left-ventricular maximal end diastolic wall thickness was > 1.5 cm in adults (Shapiro and McKenna, J Am Coll Cardiol 1983;2:437-444) or > 2 SD above the mean for a given age in children (Burch et al., J Am Coll Cardiol 1993;22:1189-1192). The clinical description of a kindred with Noonan-like/multiple giant-cell lesion syndrome was reported elsewhere (Bertola et al., Am J Med Genet 2001;98:230-234). Informed consent was obtained from all subjects included in the study.

Mutational Analysis. Genomic DNAs were isolated from peripheral blood lymphocytes (Gentra). The entire PTPNl 1 coding region (exons 1-15) was screened for mutations. For exons 2-15, PCRs were performed in a 25-μl reaction volume containing 20-80 ng genomic DNA, 1 U Amph αg Gold (Roche), 20 pmol each primer, 1.5 mM MgCl₂, 75 μM each dNTP, and lx PCR Buffer II (Roche), through use of a GeneAmp PCR System 9700 (Applied Biosystems). Exon 1 amplifications were performed using the GC-rich PCR System (Roche), according to the manufacturer's specifications. Cycling parameters were as follows: 94°C for 8 min (first denaturing step); 33 cycles of 94°C for 45 s, 54-60°C (see table 1) for 30 s, and 72°C for 45 s; and 72°C for 10 min (last extension step).

Primer pairs were designed to amplify exons, exon/intron boundaries, and short intron flanking stretches. Primer sequences, annealing temperatures, and sizes of PCR products are listed in Table 3. Mutational analysis of the amp rimers was performed by means of denaturing high-performance liquid chromatography (DHPLC), through use of the Wave DNA Fragment Analysis System (Transgenomics) at column temperatures recommended by the WaveMaker version 4.1.31 software (Transgenomics). DHPLC buffers and run conditions were as follows: buffer A (0.1M triethylammonium acetate (TEAA), 0.025% acetonitrile (ACN)), buffer B (0.1M TEAA, 25% ACN); a flow rate of 0.9 ml min; and a gradient duration of 3 min, with active clean (75%> ACN). Buffer B gradients and temperatures are reported in table 4. Positive controls — that is, PCR products expected to result in variant elution profiles — were used in all DHPLC runs.

TABLE 3 Primer Pairs and Annealing Temperatures Used to Amplify the PTPN11 Coding

Sequence and Sizes of PCR Products

Exon Primer Sequence Annealing Product

Forward (SEQ ED Reverse (SEQ LD NO) ^{Temp (}°^{C) Length (b}P NO)

60 589

2 5 6 60 405 3 7 8 60 384 Primer Sequence

4 9 10 58 447

5 11 12 60 329

6 13 14 54 282

7 15 16 56 271

8 17 18 57 350

9 19 20 56 357

10 21 22 60 284

11 23 24 60 453

12 25 26 56 250

13 27 28 60 356

14 ^a 29 30 60 259

15 31 32 60 321

GC clamps were added at the 5' end for DHPLC analysis: forward primer, 5'- CCCGCCGCCCCCGCCG-3' (SEQ ED NO:34); reverse primer, 5'- CCGCGCCCCCGCCCG-3' (SEQ ED NO:35) (product length = 290 bp).

TABLE 4 Percent Buffer B and Temperatures Used in DHPLC Analysis for PTPNl 1

Mutation Detection

EXON % BUFFER S³ TEMPERATURE(S)

Loading Initial Final (°C)

1 56 61 67 67

2 55 60 66 56, 57

3 54 59 65 57, 58

4 53 58 64 56, 57

5 51 56 62 56, 58

6 50 55 61 56, 57 EXON % BUFFER B^a TEMPERATURE(S)

Loading Initial Final (°C)

7 50 55 61 56, 57

8 51 56 62 57, 58

9 52 57 63 56, 57

10 50 55 61 57, 58

11 54 59 65 59

49 54 60 64

12 48 53 59 58, 59

13 51 56 62 59

50 55 61 60

14 52 57 63 57

49 54 60 60

15 51 56 62 56, 57

^a % buffer A - 100 - % buffer B

Heterozygous templates with previously identified mutations or single- nucleotide polymorphisms (SNPs) were used as positive controls for exons 3, 4, 7, 8, and 13. For each of the remaining exons, a synthetic template containing a single nucleotide change was constructed using the overlap extension method in a two-step PCR procedure. Wild-type and mutated PCR products were denatured together at 94°C for 5 min and were slowly cooled at room temperature, to allow heteroduplex formation. Bidirectional direct sequencing of purified PCR products (Qiagen) was performed using the ABI BigDye Terminator Sequencing Kit (Perkin Elmer) and an ABI 3700 Capillary Array Sequencer (Perkin Elmer). Sequences were analyzed by the Sequencing Analysis v.3.6.1 and AutoAssembler v.1.4.0 software packages (Perkin Elmer). Cosegregation analysis and exclusion of the mutations in control samples were performed by means of RFLP, (n = 100) DHPLC, or direct sequencing.

Genotype-phenotype correlations were performed using 2x2 contingency-table analysis. The significance threshold was set at P < 0.05. Results Spectrum of PTPNl 1 Mutations. The study population comprised 119 propositi with NS, including 70 with sporadic NS and 49 apparently unrelated families. Of the familial cases, the phenotype was linked to the NSl locus in 11. Linkage exclusion for the NSl locus was documented in four families. The small size of the remaining 34 families (typically, an affected parent and child) did not allow for linkage analysis. All subjects were of northern-European descent, except for two, one of Afro-Caribbean and one of Indian origin. DNAs from the 119 propositi with NS were screened for PTPNl 1 coding-region mutations. Exons 1-15 and flanking intron sequences were PCR amplified and were analyzed by means of DHPLC, and products with variant elution profiles were sequenced bidirectionally. PTPNl 1 mutations were identified in 54 subjects, comprising 22 different nucleotide changes (Table 5). All mutations were missense exonic changes, with the majority clustering in exons 3 and 8. The mutations cosegregated completely with the disease in all familial cases. None of the 22 different mutations was observed in at least 100 control Europeans or Americans of European descent. Parental DNAs were available for 43 of 52 (83%) individuals with sporadic NS who had PTPNl 1 mutations. Only one parent was identified as carrying a mutation - the 925A-»G transition, predicting an Ile309Val substitution. The variant profile for this mutation was not observed in >400 control chromosomes.

TABLE 5 PTPNl 1 Mutations in NS

Nucleotide Amino Acid

No. of Cases Domain Substitution Substitution

Exon 2:

124A→G 2 Thr42Ala N-SH2

Exon 3:

179G- C 2 Gly60Ala N-SH2

181G→A 1 Asρ61Asn N-SH2

184T-»G 2 Tyr62Asρ N-SH2 Nucleotide Amino Acid

No. of Cases Domain Substitution Substitution

188A→G 4 Tyr63Cys N-SH2

215C→G 1 Ala72Gly N-SH2

218C-»T 1 Thr73Ile N-SH2

228G→C 1 Glu76Asp N-SH2

236A→G 5 Gln79Arg

317A→C 3 Asp 106 Ala N-SH2/C-SH2 linker

Exon 4:

417G-»C 1 Glul39Asp C-SH2

417G→T 1 Glul39Asp C-SH2

Exon 7:

836A→G 1 Tyr279Cys PTP

844A->G 1 Ile282Val PTP

853T→C 1 Phe285Leu PTP

Exon 8:

854T→C 1 Phe285Ser PTP

922A→G 17 Asn308Asp PTP

923A→G^a 2 Asn308Ser PTP

925A→G 1 Ile309Val PTP

Exon 13:

1502G→A 1 Arg501Lys PTP

1510A→G 3 Met504Val PTP ^a Affected members of one family segregating the 923A->G change (Asn308Ser) exhibited the Noonan-like/multiple giant-cell lesion condition.

The A→G transition at position 922 in exon 8, predicting the Asn308Asp substitution within the PTP domain, was the most common mutation, accounting for one-third of the total. Of note, the Asn308Asp mutation was identified in the large three-generation family that was used to originally establish linkage to the NSl locus (Jamieson et al., Nat Genet 1994;8:357-360; van der Burgt et al., Am J Med Genet 1994;94:46-51) (Figure 5). The two occurrences of the adjacent 923A→G mutation, predicting an Asn308Ser change, indicate that codon 308 represents a hotspot for NS. Eight additional mutations were, found recurrently. Among them, the 182A→G (AspβlGly), 188A→G (Tyr63Cys), and 236A→G (Gln79Arg) substitutions in exon 3, as well as the 1510A- G (Met504Val) change in exon 13, have previously been reported as disease-causing mutations (Tartaglia et al., Nat Genet 2001;29:465-468). Interestingly, 90% of mutational events involved amino acid residues located in the N-SH2 and PTP functional domains. The exceptions were the 317A-»C transversion, observed in two individuals with sporadic NS and in one family, predicting an Aspl06Ala substitution in the peptide linking the N-SH2 and C- SH2 domains, and the 417G-»C and 417G-s>T changes, both predicting a Glul39Asp substitution within the C-SH2 domain.

On the basis of the secondary structure of PTPNl 1 in the inactive conformation determined by Hof et al. (Cell 1998:92:441-450), the N-SH2 and PTP mutations were noted to cluster dramatically in specific regions of those domains. Of the 10 N-SH2 substitutions, 5 were positioned in the D'E loop and flanking βD' and βE strands (residues 57-65), and 4 were in the βF strand and αB helix (residues 69- 84). The nine PTP mutations were restricted to the DB loop (residues 277-288; n = 4), βC strand (residues 303-311; n = 3), and HI loop (residues 499-507; n - 2). Most strikingly, all N-SH2 and PTP mutations except the Thr42Ala substitution involved residues located in or close to the N-SH2/PTP interacting surface.

Fifteen sequence variants representing polymorphisms were observed in subjects with NS, unaffected family members, and control individuals (Table 6). These changes included 13 intronic SNPs, a single-base deletion within the 5'-UTR, and a synonymous change in exon 3.

TABLE 6 Polymorphisms in the PTPN11 Gene

Location Nucleotide Change Position of SEQ ID NO:33

Exon 1(5' UTR) -140delG^a 3123451

Intron 1 +25G→C 3123629 Intron 1 +54C→A 3123658

Exon 3 255C→T (His85) 3194548

Intron 4 +12G→C 3194637

Intron 4 +78A-»G 3194703

Intron 7 -21C→T 3221743

Intron 7 -30T→C 3221734

Intron 7 -32A→C 3221732

Intron 7 -35A→C 3221729

Intron 7 -132T - C 3221632-

Intron 9 -9C→A 3229178

Intron 10 -63G→A 3230525

Intron 13 +26G- A 3233314

Intron 15 +40T→C 3248949

Position refers to the A of the ATG initiator codon of SEQ ID NO:l, corresponding to nucleotide 3123591 in SEQ ID NO:33.

Genetic Heterogeneity in NS. PTPNl 1 mutations were detected in the 11 families for which the disorder appeared to be linked to the NSl locus, including the original large family described by Jamieson et al. (1994). As anticipated, no PTPNl 1 sequence change was observed in the four families for which linkage to NSl had been excluded. For the entire study group of 119, PTPNl 1 mutations were observed in 45%, a slightly lower incidence than had been seen in a small cohort (Tartaglia et al., Nature Genet 2001;29:465-468). PTPNl 1 mutations were detected in 59%) of individuals with familial NS, whereas such defects were observed in only 37%o of individuals with sporadic NS. This statistically significant (P < 0.02) difference in PTPNl 1 mutation prevalence suggests that the additional gene or genes responsible for NS engender incomplete penetrance or have greater adverse affects on fertility than does PTPNl 1.

Genotype-Phenotype Correlation. Because of the clinical heterogeneity observed in NS, we investigated possible associations between genotype and phenotype. The distribution of several major clinical features of NS, in subjects with and without mutations in PTPN11, is shown in Table 7. A statistically significant association with pulmonic stenosis was found in the group with PTPNl 1 mutations (70.6%> vs. 46.2%; P = 0.008). In contrast, a statistically significantly lower incidence of HCM was observed in this group (5.9%) vs. 26.2%o; p = 0.004). There was no significant difference in the prevalence of atrial and/or ventricular septal defects or other congenital heart malformations between the groups with and without PTPNl 1 mutations. Similarly, there was no difference in the rates of short stature, pectus deformities, cryptorchidism, or emoUment in special education (as a marker of developmental delay).

TABLE 7 Clinical Features in Subjects with NS with and without PTPNl 1 Mutations

NO./TOTAL (%) OF SUBJECTS With PTPN11 Without PTPN11

Clinical Feature Mutation Mutation p^a

Cardiac defects:

HCM 3/51 (5.9) 17/65 (26.1) .004

Pulmonic stenosis 36/51 (70.6) 30/65 (46.2) .008

Septal defects 6/50/(12.0) 11/63 (17.5) NS

Short stature 39/51 (76.5) 45/64 (70.3) NS

Special education 11/46 (23.9) 21/59 (35.6) NS

Pectus deformities 39/50 (78.0) 46/61 (75.4) NS

Cryptorchidism 26/31 (83.9) 25/35 (71.4) NS ^a "NS" indicates a difference that is not statistically significant.

The clinical manifestations of NS were compared between the cohort with N-SH2 mutations and the cohort with PTP mutations. Although this analysis had less statistical power, owing to sample size, no significant differences were identified. The phenotype observed in subjects with the common Asn308Asp substitution (n = 17) was not qualitatively different from the phenotype in subjects with other mutations, except for the fact that no subject carrying the Asn308Asp change was enrolled in special education.

We identified an A-»G transition at position 923 (Asn308Ser) in a family with typical features of NS associated with multiple giant-cell lesions in bone (Bertola et al., Am J Med Genet 2001,98:230-234). This mutation cosegregated perfectly with this phenotype (Figure 6). One family member (III-3) had a typical NS phenotype (i.e., pulmonic stenosis, cryptorchidism, short stature, and distinctive face and chest deformity) and exhibited asymptomatic lesions in the right ramus of the mandible and in the maxilla, as well as osteolytic lesions in both humeri and in the left femur. Similarly, his affected sister (III-2) had multiple lesions in the mandible. Serum calcium, phosphate, and PTH levels were normal in both subjects. Their mother (II-2) had typical NS features but did not have any bone lesions. The same Asn308Ser mutation was observed in another family with NS that had no known bony involvement.

Discussion

In our present analysis of a large cohort with sporadic and familial cases of NS, we estimated the PTPNl 1 mutation prevalence to be 45%. This is quite similar to the rate of 50%> observed in a small cohort with NS. In the present study, however, DHPLC was used to screen the PTPNl 1 coding exons. Since this method is purported to have a sensitivity of 96%>-100% under ideal conditions (Xiao and Oefner, Hum Mut 2001;17:439-474), the true prevalence of point mutations in this cohort could be slightly higher. There may also be other types of molecular lesions that might cause NS, such as large intragenic deletions, changes in the 3'UTR, or promoter defects. Lastly, the prevalence detected in any NS cohort would be sensitive to the composition of sporadic and familial cases. With those issues stipulated, the contribution of PTPNl 1 mutations to the etiology of NS appears to be approximately 50%). When these results are combined with our previous work, we have now identified 78 unrelated individuals with NS who have mutations in PTPNl 1. All mutations are conserved among the vertebrate PTPNl 1 orthologs. Most altered residues are also conserved among the vertebrate SHP-1 proteins.

As described herein, we have found that the NS-causing PTPNl 1 defects result in gain-of-function effects on PTPNl 1. This was supported by energetics-based structural analysis of two mutants (Tartaglia et al., Nat Genet 2001;29:456-468) and by the observation that two NS mutant alleles closely resembled two engineered PTPNl 1 mutants with increased phosphatase activity in vitro and inductive effects in Xenopus animal caps (O'Reilly et al., Mol Cell Biol 2000;20:299-311). Since we have not identified any nonsense, frameshift, or splicing defect among 78 PTPNl 1 mutations, it seems highly unlikely that PTPNl 1 haploinsufficiency results in the NS phenotype.

The distribution of the altered amino acid residues in PTPNl 1 has a nonrandom pattern (Table 7). The vast majority of the NS mutations clustered in the N-SH2 and PTP functional domains but were not restricted to those domains, as was seen previously. The N-SH2 domain interacts with the PTP domain and binds to phosphotyrosyl-containing targets on activated receptors or docking proteins, using two separate sites. These sites show negative cooperativity, so that N-SH2 can work as intramolecular switch to control PTPNl 1 catalytic activity. In the inactive state, the N-SH2 and PTP domains share a broad interaction surface. More precisely, the N- SH2 D'E loop and flanking βD' and βE strands closely interact with the catalytic cleft, blocking the PTP active site. Crystallographic data on PTPNl 1 in the inactive conformation revealed a complex interdomain hydrogen-bonding network — involving Asn58, Gly60, Asp61, Cys459, and Gln506 — that stabilizes the protein (Hof et al., Cell 1998;92:441-450). Numerous polar interactions between N-SH2 residues located in strands βF and β A, helix αB, and residues of the PTP domain further stabilize the inactive conformation. Significantly, most of the residues mutated in NS are either directly involved in these interdomain interactions (i.e., Gly60, Aspόl, Ala72, Glu76, and Gln79) or in close spatial proximity to them (i.e., Tyr62, Tyr63, Thr73, Tyr279, Ile282, Phe285, Asn308, Ile309, Arg501, and Met504). This distribution of molecular lesions suggests that the pathogenetic mechanism in NS involves altered N-SH2/PTP interactions that destabilize the inactive conformation without altering PTPNll's catalytic capability. Consistent with this view, no mutation altered Cys459 (the residue essential for nucleophilic attack), the PTP signature motif (positions 457- 467), or the TrpProAsp loop (positions 423-425), which are all essential for phosphatase activity.

Three of the NS mutations affected residues outside of the interacting regions of the N-SH2 and PTP domains. One recurrent mutation affected Asp 106, which is located in the linker stretch connecting the N-SH2 and C-SH2 domains. Although functional studies are required to understand the functional significance of the Asp-to-Ala substitution, we hypothesize that this mutation might alter the flexibility of the N-SH2 domain, thus inhibiting the N-SH2/PTP interaction. Two mutated residues, Thr42 (N-SH2 domain) and Glul39 (C-SH2 domain), are spatially far from the N-SH2/PTP interaction surfaces. In contrast to the other mutated residues, Thr42 and Glul39 are implicated in the intermolecular interactions of the SH2 domains with phosphotyrosyl-containing peptides (Lee et al., Structure 1994;2:423-438; Huyer and Ramachandran, Biochemistry 1998;37:2741-2747). Specifically, Thr42 directly interacts with the tyrosine phosphate, and Glul39 is adjacent to Argl38 and Serl40, which form hydrogen bonds to that phosphate. The phenotype of the subjects bearing these mutations was typical for NS.

Previously, the penetrance of NS had not been addressed in a systematic fashion. Two lines of evidence now suggest that NS caused by PTPNl 1 mutations is almost completely penetrant. First, we analyzed 11 families, including the large kindred first described by van der Burgt et al. (Am J Med Genet 1994;94:46- 51), for which significant or suggestive linkage to the NSl locus had been established (Jamieson et al., Nat Genet 1994;8:357-360). In each instance, a PTPN11 mutation was identified that was inherited by all of the affected individuals in the family but by none of the unaffected ones. Analysis of small kindreds harboring PTPNl 1 mutations revealed the same consistent pattern. Second, we genotyped a high percentage (83%) of the unaffected parents for defects discovered in their offspring with apparently sporadic NS. In only one instance was a mutation identified. Although it is possible that inclusion of "milder" cases of NS might have uncovered some instances of incomplete penetrance, the strict criteria for NS employed in the present study identified a cohort with almost 100%) penetrance.

We observed a statistically significantly higher incidence of pulmonic stenosis among subjects with NS inheriting PTPNl 1 mutations. Experimental evidence with mice indicates that epidermal growth factor (EGF) signaling is important for semilunar valve development and that PTPNl 1 is a component of the EGF-mediated signal transduction pathway (Qu et al., PNAS 1999;96:8528-8533; Chen et al., Nat Genet 2000;24:296-299). A proportion of mice that are homozygous for a hypomorphic Egfr allele exhibit thickened aortic and pulmonary valve leaflets because of an increased number of mesenchymal cells. Coinheritance of a Ptpnll knock-out allele in heterozygosity results in a higher prevalence and increased severity of those valve abnormalities. These findings implicate PTPNl 1 in aspects of EGF-mediated semilunar valvulogenesis, such as mesenchymal transformation and proliferation, as well as leaflet remodeling. Finally, we demonstrated the cosegregation of a 923A-»G PTPNl 1 mutation in a family inheriting the Noonan-like/multiple giant-cell lesion syndrome. Since a 923A-»G mutation was also identified in an unrelated kindred with classic NS, additional genetic factors or events may be necessary to result in the proliferation of these giant cells. Only a single family with this rare phenotype was available for genotyping. Although the Noonan-like/multiple giant-cell lesion syndrome was introduced as a distinct entity characterized by the association of some cardinal features of NS with giant-cell lesions of bone and soft tissues (Cohen et al., Syndrome Ident 1974;2:14-17; Cohen and Gorlin, Am J Med Genet 1991;40159-166), it is now a part of the NS spectrum.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all values are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS :

1. A method for diagnosing Noonan syndrome in a subject, which method comprises detecting a mutation in a protein tyrosine phosphatase 11 (PTPNl 1) gene in the subject, wherein the mutation results in increased PTPNl 1 expression or activity as compared to a control.

2. The method of claim 1, wherein the mutation is a member of the group consisting of a missense mutation, a deletion, an insertion, and both a deletion and an insertion.

3. The method of claim 1, wherein the mutation is in a coding region of the gene.

4. The method of claim 3, wherein the mutation in the coding region results in a mutation in the PTPNl 1 protein.

5. The method of claim 4, wherein the mutation in the PTPNl 1 protein is in an src-homology 2 (SH2) domain.

6. The method of claim 5, wherein the mutation in the PTPNl 1 protein is an amino acid substitution selected from the group consisting of a) a G to A substitution at position 60 of SEQ ED NO:2; b) a D to N substitution at position 61 of SEQ ID NO:2; c) a D to G substitution at position 61 of SEQ ED NO:2; d) a Y to D substitution at position 62 of SEQ ED NO :2; e) a Y to C substitution at position 63 of SEQ LD NO :2; f) an A to S substitution at position 72 of SEQ ED NO:2; g) an A to G substitution at position 72 of SEQ ID NO:2; h) an E to D substitution at position 76 of SEQ ED NO:2; and i) a Q to R substitution at position 79 of SEQ LD NO:2.

7. The method of claim 5, wherein the mutation in the PTPNl 1 protein is an amino acid substitution selected from the group consisting of a) a T to A substitution at position 42 of SEQ ED NO :2; b) a T to I substitution at position 73 of SEQ LD NO:2; and c) an E to D substitution at position 139 of SEQ ED NO:2.

8. The method of claim 5, wherein the mutation in the PTPNl 1 protein is a G to V substitution at position 60 and a deletion of D at position 61 of SEQ ED NO:2.

9. The method of claim 5, wherein the mutation in the PTPNl 1 gene is selected from the group consisting of a) a G to C substitution at position 179 of SEQ ED NO: 1 ; b) a G to A substitution at position 181 of SEQ ED NO: 1 ; c) an A to G substitution at position 182 of SEQ ED NO: 1 ; d) a T to G substitution at position 184 of SEQ ED NO : 1 ; e) an A to G substitution at position 188 of SEQ ED NO: 1 ; f) a G to T substitution at position 214 of SEQ LD NO: 1 ; g) a C to G substitution at position 215 of SEQ ED NO: 1 ; h) an G to C substitution at position 228 of SEQ ED NO: 1 ; and i) an A to G substitution at position 236 of SEQ ED NO: 1.

10. The method of claim 5, wherein the mutation in the PTPNl 1 gene is selected from the group consisting of a) an A to G substitution at position 124 of SEQ ED NO: 1 ; b) a C to T substitution at position 218 of SEQ ED NO: 1 ; c) a G to C substitution at position 417 of SEQ LD NO: 1 ; and d) a G to T substitution at position 417 of SEQ LD NO : 1.

11. The method of claim 10, wherein the mutation in the PTPNl 1 gene is a G to T substitution at position 179 and a deletion of positions 180-182 of SEQ ED NO:l.

12. The method of claim 4, wherein the mutation in the PTPNl 1 protein is a linker domain connecting a first SH2 domain to a second SH2 domain.

13. The method of claim 12, wherein the mutation is a D to A amino acid substitution at position 106 of SEQ LD NO:2.

14. The method of claim 12, wherein the mutation in the PTPNl 1 gene is an A to C substitution at position 317 of SEQ ED NO:l.

15. The method of claim 4, wherein the mutation in the PTPNl 1 protein is in a protein tyrosine phosphatase (PTP) domain.

16. The method of claim 15, wherein the mutation is an amino acid substitution selected from the group consisting of a) an Y to C substitution at position 279 of SEQ LD NO:2; b) an I to V substitution at position 282 of SEQ ED NO:2; c) an F to L substitution at position 285 of SEQ ED NO:2; d) an F to S substitution at position 285 of SEQ LD NO:2; e) an N to D substitution at position 308 of SEQ LD NO:2; f) an N to S substitution at position 308 of SEQ TD NO:2; g) an I to V substitution at position 309 of SEQ ED NO:2; h) an R to K substitution at position 501 of SEQ ED NO:2; and i) an M to V substitution at position 504 of SEQ LD NO:2.

17. The method of claim 15, wherein the mutation is an amino acid substitution selected from the group consisting of a) a G to S substitution at position 268 of SEQ LD NO:2; b) an N to T substitution at position 308 of SEQ LD NO:2; c) an P to S substitution at position 491 of SEQ ED NO:2; and d) an S to L substitution at position 502 of SEQ LD NO:2.

18. The method of claim 15, wherein the mutation in the PTPNl 1 gene is selected from the group consisting of a) an A to G substitution at position 836 of SEQ LD NO: 1 ; b) an A to G substitution at position 844 of SEQ LD NO: 1 ; c) a T to C substitution at position 853 of SEQ LD NO: 1 ; d) a T to C substitution at position 854 of SEQ LD NO: 1 ; e) an A to G substitution at position 922 of SEQ ED NO: 1 ; f) an A to G substitution at position 923 of SEQ ED NO: 1 ; g) an A to G substitution at position 925 of SEQ TD NO: 1 ; h) a G to A substitution at position 1502 of SEQ ED NO: 1 ; and i) an A to G substitution at position 1510 of SEQ ED NO: 1.

19. The method of claim 12, wherein the mutation in the PTPNl 1 gene is selected from the group consisting of a) a G to A substitution at position 802 of SEQ ED NO: 1 ; h) an A to C substitution at position 923 of SEQ ED NO: 1 ; j) a C to T substitution at position 1471 of SEQ ED NO : 1 ; and

1) a C to T substitution at position 1505 of SEQ LD NO:l.

20. A kit for diagnosing Noonan syndrome, comprising an oligonucleotide that specifically hybridizes to or adjacent to a site of mutation of a PTPNl 1 gene that results in increased activity of a PTPNl 1 protein encoded by the gene; and instructions for use.

21. The kit of claim 20, wherein the site of mutation comprises a nucleotide selected from the group consisting of nucleotides 179, 181, 182, 184, 188, 214, 215, 228, 236, 317, 836, 844, 853, 854, 922, 923, 925, 1502, and 1510 of SEQ ED NO:l.

22. The kit of claim 20, wherein the site of mutation comprises a nucleotide selected from the group consisting of nucleotides 124, 180, 218, 417, 802, 1403, 1471, and 1505 of SEQ TD NO:l.

23. The kit of claim 20, comprising at least one probe comprising the site of mutation.

24. The kit of claim 20, comprising a first oligonucleotide primer comprising at least 15 consecutive nucleotides of SEQ TD NO:33, and a second oligonucleotide primer comprising at least 15 consecutive nucleotides of a sequence complementary to SEQ ID NO: 33.

25. The kit of claim 20, comprising a first primer comprising a nucleotide sequence selected from the group consisting of SEQ ED NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31, and a second primer selected from the group consisting of SEQ LD NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32.

26. A kit for diagnosing Noonan syndrome, comprising an antibody that specifically recognizes a mutation in a PTPNl 1 protein; and instructions for use.

27. The kit of claim 26, wherein the mutation results in an increased activity as compared to a PTPNl 1 protein having the amino acid sequence of SEQ ED NO:2.

28. The kit of claim 26, wherein the mutation is in an SH2 domain.

29. The kit of claim 28, wherein the mutation is selected from the group consisting of a) a G to A substitution at position 60 of SEQ ED NO:2; b) a D to N substitution at position 61 of SEQ ED NO:2; c) a D to G substitution at position 61 of SEQ LD NO:2; d) a Y to D substitution at position 62 of SEQ ED NO:2; e) a Y to C substitution at position 63 of SEQ ED NO:2; f) an A to S substitution at position 72 of SEQ ED NO:2; g) an A to G substitution at position 72 of SEQ ED NO:2; h) an E to D substitution at position 76 of SEQ LD NO:2; and i) a Q to R substitution at position 79 of SEQ LD NO:2.

30. The kit of claim 28, wherein the mutation is selected from the group consisting of a) a T to A substitution at position 42 of SEQ ED NO:2; b) a G to V substitution at position 60 in combination with a deletion of position 61 of SEQ TD NO:2; b) a T to I substitution at position 73 of SEQ TD NO:2; and c) an E to D substitution at position 139 of SEQ LD NO:2.

31. The kit of claim 26, wherein the mutation is in a linker domain connecting a first SH2 domain to a second SH2 domain.

32. The kit of claim 31, wherein the mutation is a D to A substitution at position 106 of SEQ ED NO:2.

33. The kit of claim 26, wherein the mutation is in a PTP domain.

34. The kit of claim 33, wherein the mutation is selected from the group consisting of a) an Y to C substitution at position 279 of SEQ ED NO:2; b) an I to V substitution at position 282 of SEQ ED NO:2; c) an F to L substitution at position 285 of SEQ ED NO:2; d) an F to S substitution at position 285 of SEQ ED NO:2; e) an N to D substitution at position 308 of SEQ LD NO:2; f) an N to S substitution at position 308 of SEQ ED NO:2; g) an I to V substitution at position 309 of SEQ ED NO:2; h) an R to K substitution at position 501 of SEQ LD NO:2; and i) an M to V substitution at position 504 of SEQ ED NO:2.

35. The kit of claim 33, wherein the mutation is selected from the group consisting of a) a G to S substitution at position 268 of SEQ TD NO:2; b) an N to T substitution at position 308 of SEQ ED NO:2; c) an P to S substitution at position 491 of SEQ LD NO:2; and d) an S to L substitution at position 502 of SEQ D NO:2.

36. A method for diagnosing Noonan syndrome in a subject, which method comprises assessing the level of expression or activity of a PTPNl 1 protein in the test subject and comparing it to the level of expression or activity in a control subject, wherein an increased expression or basal activity of the PTPNl 1 protein in the test subject compared to the control subject is indicative of Noonan syndrome.

37. The method of claim 36, wherein the level of expression is assessed by determining the amount of mRNA that encodes the PTPNl 1 protein in a biological sample.

38. The method of claim 36, wherein the level of expression of PTPNl 1 is assessed by determining the concentration of PTPNl 1 protein in a biological sample.

39. The method of claim 36, wherein the level of activity is assessed by determining the level of phosphatase activity of the protein.

40. A method for treating Noonan syndrome in a patient, which method comprises administering to the patient in need of such treatment an effective amount of an agent that modulates the expression or activity of a PTPNl 1 protein, in association with a pharmaceutically acceptable carrier.

41. The method of claim 40, wherein the PTPNl 1 protein comprises the amino acid sequence of SEQ LD NO:2.

42. The method of claim 40, wherein the agent is a PTPN11 antisense nucleic acid.

43. The method of claim 42, wherein the antisense nucleic acid hybridizes to a segment of SEQ TD NO:l comprising at least one nucleotide substitution selected from the group consisting of a) a G to C substitution at position 179 of SEQ LD NO : 1 ; b) a G to A substitution at position 181 of SEQ ED NO: 1 ; c) an A to G substitution at position 182 of SEQ ED NO : 1 ; d) a T to G substitution at position 184 of SEQ ED NO: 1 ; e) an A to G substitution at position 188 of SEQ ED NO: 1 ; f) a G to T substitution at position 214 of SEQ ED NO:l; g) a C to G substitution at position 215 of SEQ LD NO: 1 ; h) an G to C substitution at position 228 of SEQ LD NO: 1 ; i) an A to G substitution at position 236 of SEQ ED NO: 1 ; and complementary segments thereof.

44. The method of claim 42, wherein the antisense nucleic acid hybridizes to a segment of SEQ TD NO:l comprising at least one nucleotide substitution selected from the group consisting of a) an A to G substitution at position 124 of SEQ ED NO: 1 ; b) a C to T substitution at position 218 of SEQ LD NO: 1 ; c) a G to C substitution at position 417 of SEQ TD NO:l; d) a G to T substitution at position 417 of SEQ ED NO: 1 ; and complementary segments thereof.

45. The method of claim 42, wherein the antisense nucleic acid hybridizes to a segment of SEQ ED NO:l comprising a G to T substitution at position 179 and a deletion of positions 180-182.

46. The method of claim 42, wherein the antisense nucleic acid hybridizes to a segment of SEQ TD NO:l comprising at least one nucleotide substitution selected from the group consisting of a) an A to G substitution at position 836 of SEQ ED NO: 1 ; b) an A to G substitution at position 844 of SEQ ED NO: 1 ; c) a T to C substitution at position 853 of SEQ LD NO: 1 ; d) a T to C substitution at position 854 of SEQ ED NO: 1 ; e) an A to G substitution at position 922 of SEQ ED NO: 1 ; f) an A to G substitution at position 923 of SEQ LD NO: 1 ; g) an A to G substitution at position 925 of SEQ LD NO: 1 ; h) a G to A substitution at position 1502 of SEQ LD NO: 1 ; i) an A to G substitution at position 1510 of SEQ ED NO: 1 ; and complementary sequences thereof.

47. The method of claim 42, wherein the antisense nucleic acid hybridizes to a segment of SEQ LD NO:l comprising at least one nucleotide substitution selected from the group consisting of a) an G to A substitution at position 802 of SEQ ED NO: 1 ; h) an A to C substitution at position 923 of SEQ ED NO: 1 ; j) a C to T substitution at position 1471 of SEQ ED NO: 1 ; and

1) a C to T substitution at position 1505 of SEQ ED NO: 1. and complementary segments thereof.

48. The method of claim 40, wherein the agent inhibits PTPNl 1 activity by blocking a PTP domain.

49. The method of claim 47, wherein the agent is an anti-PTPNll inhibitory antibody.

50. The method of claim 49, wherein the antibody specifically recognizes a PTPNl 1 amino acid sequence comprising mutation selected from the group consisting of: a) a G to A substitution at position 60 of SEQ LD NO:2; b) a D to N substitution at position 61 of SEQ ED NO:2; c) a D to G substitution at position 61 of SEQ LD NO :2; d) a Y to D substitution at position 62 of SEQ ED NO:2; e) a Y to C substitution at position 63 of SEQ LD NO:2; f) an A to S substitution at position 72 of SEQ ED NO:2; g) an A to G substitution at position 72 of SEQ ED NO:2; h) an E to D substitution at position 76 of SEQ ED NO:2; i) a Q to R substitution at position 79 of SEQ LD NO :2; j) a D to A substitution at position 106 of SEQ ED NO:2; k) a Y to C substitution at position 279 of SEQ ED NO:2; 1) an I to V substitution at position 282 of SEQ LD NO:2; m) an F to L substitution at position 285 of SEQ LD NO:2; n) an F to S substitution at position 285 of SEQ ED NO:2; o) an N to D substitution at position 308 of SEQ ED NO:2; p) an N to S substitution at position 308 of SEQ LD NO:2; q) an I to V substitution at position 309 of SEQ TD NO:2; r) an R to K substitution at position 501 of SEQ ED NO:2; and s) an M to V substitution at position 504 of SEQ ED NO:2.

51. The method of claim 49, wherein the antibody specifically recognizes a PTPNl 1 amino acid sequence comprising an amino acid substitution selected from the group consisting of: a) a T to A substitution at position 42 of SEQ ED NO:2; b) a G toV substitution at position 60 in combination with a deletion of position 61 of SEQ ED NO:2; c) a T to I substitution at position 73 of SEQ ED NO:2; d) an E to D substitution at position 139 of SEQ ED NO:2; e) a G to S substitution at position 268 of SEQ ED NO:2; f) an N to T substitution at position 308 of SEQ TD NO:2; g) an P to S substitution at position 491 of SEQ ED NO:2; and h) an S to L substitution at position 502 of SEQ LD NO:2.

52. An isolated PTPNl 1 variant comprising a mutation resulting in increased level of PTPNl 1 activity.

53. The isolated PTPNl 1 variant of claim 52, comprising an amino acid substitution in a region selected from the group consisting of an N-SH2 domain, C- SH2 domain, PTP domain, and a region between an N-SH2 and a C-SH2 domain.

54. The isolated PTPNl 1 variant of claim 53, wherein the amino acid substitution is selected from the group consisting of a) a G to A substitution at position 60 of SEQ LD NO:2; b) a D to N substitution at position 61 of SEQ ED NO:2; c) a D to G substitution at position 61 of SEQ LD NO:2; d) a Y to D substitution at position 62 of SEQ LD NO:2; e) a Y to C substitution at position 63 of SEQ LD NO:2; f) an A to S substitution at position 72 of SEQ ED NO:2; g) an A to G substitution at position 72 of SEQ ED NO:2; h) an E to D substitution at position 76 of SEQ LD NO:2; i) a Q to R substitution at position 79 of SEQ ID NO:2; j) a D to A substitution at position 106 of SEQ ED NO:2; k) an Y to C substitution at position 279 of SEQ LD NO:2;

1) an I to V substitution at position 282 of SEQ ED NO:2; m) an F to L substitution at position 285 of SEQ LD NO:2; n) an F to S substitution at position 285 of SEQ ED NO:2; o) an N to D substitution at position 308 of SEQ LD NO:2; p) an N to S substitution at position 308 of SEQ LD NO:2; q) an I to V substitution at position 309 of SEQ LD NO:2; r) an R to K substitution at position 501 of SEQ LD NO:2; and s) an M to V substitution at position 504 of SEQ LD NO:2.

55. The isolated PTPNl 1 variant of claim 53, wherein the amino acid substitution is selected from the group consisting of a) a T to A substitution at position 42 of SEQ ED NO:2; b) a G toV substitution at position 60 in combination with a deletion of position 61 of SEQ ED NO:2; c) a T to I substitution at position 73 of SEQ ED NO:2; d) an E to D substitution at position 139 of SEQ LD NO:2; e) a G to S substitution at position 268 of SEQ LD NO:2; f) an N to T substitution at position 308 of SEQ LD NO:2; g) an P to S substitution at position 491 of SEQ ED NO:2; and h) an S to L substitution at position 502 of SEQ ED NO:2.

56. An isolated cell comprising a vector, which vector comprises a nucleic acid encoding the PTPNl 1 variant of claim 54, operatively associated with an expression control sequence.

57. The cell of claim 56, selected from a prokaryotic cell and an eukaryotic cell.

58. An isolated cell comprising a vector, which vector comprises a nucleic acid encoding the PTPNl 1 variant of claim 55, operatively associated with an expression control sequence.

59. The cell of claim 58, selected from a prokaryotic cell and an eukaryotic cell.

60. An isolated nucleic acid encoding the PTPNl 1 variant of claim 54.

61. An isolated oligonucleotide which specifically hybridizes to the nucleic acid of claim 60.

62. An isolated nucleic acid encoding the PTPNl 1 variant of claim 55.

63. An isolated oligonucleotide which specifically hybridizes to the nucleic acid of claim 62.