Compositions and methods of diagnosis and treatment of lymphatic system disorders

Abstract

To provide compositions and methods for diagnosis and treatment of lymphatic vessel anomaly.SOLUTION: Disclosed is a method for diagnosing a lymphatic anomaly in a human patient comprising the steps of: a) obtaining a biological sample comprising a nucleic acid from the patient; and b) assaying the nucleic acid to determine whether i) a single nucleotide variant (SNV) in one or more of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF and CBL is present or ii) an SNV in linkage disequilibrium with an SNV in one or more of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF and CBL is present; and c) diagnosing the patient as lymphatic anomaly when an SNV of i) or ii) is present.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 728,444, filed September 7, 2018, the entire contents of which are incorporated herein by reference as if fully set forth.

[0002] The present invention relates to the fields of genetics, personalized medicine, and lymphatic malformations, and in particular provides novel genetic targets and therapeutic regimens to ameliorate symptoms associated with lymphangiomatosis and other generalized lymphatic malformations (GLA). [Background technology]

[0003] Several publications and patent documents are cited herein to describe the state of the art to which the present invention pertains, and these references are incorporated herein by reference as if set forth in full.

[0004] The lymphatic system plays a vital role in maintaining fluid circulation, protecting the body from disease, and absorbing dietary fat in the small intestine (1). Complex lymphatic disorders are characterized by abnormal formation of lymphatic vessels and tissue overgrowth. Patients often present with overlapping symptoms, which can lead to severe pulmonary disease (2, 3). Examples of lymphatic disorders include generalized lymphatic anomalies (GLA), lymphangiectasia, and bile collection (pericardial, pleural, and peritoneal). The study of complex lymphatic disorders is hampered by inconsistent classification and nomenclature, as diagnostic challenges are significant (3-6). Although the molecular genetic pathogenesis of complex lymphatic disorders is poorly understood, congenital malformations of the lymphatic system appear to have associated genetic etiologies (7-9). Indeed, both germline and somatic mutations have been identified in genes clustered in the PI3K / mTOR and Ras / MAPK pathways (1, 8).

[0005] Disruptions or abnormalities in the PI3K / mTOR and Ras / MAPK signaling pathways have been shown to impair normal expansion and remodeling in establishing mature lymphatic networks, and such disruptions are associated with lymphatic vascular diseases. Gain of function mutations in AKT1 and PIK3CA, which result in increased mammalian target of rapamycin complex 1 (mTORC1) activity, have been identified in patients with lymphatic malformations that are part of syndromes such as Proteus syndrome (OMIM 176920), CLOVES syndrome (OMIM 612918), and Klippel-Trenaunay-Weber syndrome (OMIM 149000) (9-11). Mutations in KRAS, HRAS, RAF1, PTPN11, SOS1, and RASA1 lead to deregulated RAS pathway activity, resulting in lymphedema and lymphangiectasia in diseases such as Noonan syndrome (OMIM 163950), Costello syndrome (OMIM 218040), myocardial infarction syndrome (OMIM 115150), and capillary malformation-arteriovenous malformation (CM-AVM) syndrome (OMIM 608354) ( 12 – 17 ).

[0006] However, there is a lack of genetic biomarkers to identify patients with lymphatic diseases and disorders, such as lymphangiomatosis / lymphangiectasia (LAM), generalized lymphatic malformation (GLA), and pleural effusion, as well as therapies targeting the genetic markers associated with these diseases. Summary of the Invention [Means for solving the problem]

[0007] Thus, in one embodiment of the present invention, a method of diagnosing a lymphatic abnormality in a human patient is provided. An exemplary method includes obtaining a biological sample comprising nucleic acid from the patient, assaying the nucleic acid to determine whether i) one or more single nucleotide variants (SNVs) in PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF are present, or ii) an SNV in linkage disequilibrium with one or more SNVs in PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF is present, and diagnosing the patient as having a lymphatic abnormality if the SNV in i) or ii) is present. In another aspect, a method of diagnosing a lymphatic abnormality in a human patient includes obtaining genotype sequence information from the human patient; determining from the sequence information whether i) one or more single nucleotide variants (SNVs) in PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF are present, or ii) an SNV that is in linkage disequilibrium with one or more SNVs among PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF is present; and diagnosing the patient with a lymphatic abnormality if the SNV in i) or ii) is present.

[0008] The present invention also provides a method of treating a lymphatic disorder in a human patient. An exemplary method includes obtaining a biological sample comprising nucleic acid from the patient, assaying the nucleic acid to determine whether i) a single nucleotide variant (SANV) is present in one or more of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL, or ii) an SNV is present that is in linkage disequilibrium with one or more of the SNVs in PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL, and administering to a patient identified as having one or more SNVs of i) or ii), one or more agents suitable for treating said lymphatic disorder, thereby treating the lymphatic disorder. In an alternative embodiment of the method, the method comprises obtaining genotype information from a patient, assaying to determine whether i) there is a single nucleotide variant (SNV) in one or more of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL, or ii) there is an SNV in linkage disequilibrium with one or more of the single nucleotide variants in PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL, and administering to a patient identified as having one or more SNVs of i) or ii) one or more agents suitable for treating said lymphatic abnormality, thereby treating the lymphatic abnormality. In an alternative embodiment of the method, genotype information is obtained from a patient.

[0009] In certain embodiments, the lymphatic abnormality is characterized by abnormal formation of lymphatic vessels and / or tissue overgrowth. In other embodiments, the lymphatic abnormality is lymphangiomatosis (LAM). In another embodiment, the lymphatic abnormality is generalized lymphatic abnormality (GLA). The lymphatic abnormality may be characterized by chylous effusions, including pericardial, pleural, or peritoneal fluid.

[0010] The diagnostic method may further comprise generating a report identifying the SNV after detection in the biological sample.The therapeutic method described above may further comprise generating a report identifying a recommended treatment(s) for the lymphatic abnormality based on the SNV identified by the method.

[0011] In some embodiments, the agents administered to the SNV positive subject are: 1) a MEK / ERK inhibitor; 2) an agent / inhibitor listed in Tables 1 and 2; 3) a combination of a MEK / ERK inhibitor with one or more agents / inhibitors listed in Tables 1 and 2, and / or 4) a combination of 1) an mTOR inhibitor and / or a PIK3K inhibitor, and 2) one or more MEK / ERK inhibitors. In yet another embodiment, the diagnostic methods described herein can further comprise administering to the diagnosed patient an effective amount of one or more agents suitable for treating said lymphatic abnormality.

[0012] In certain embodiments of the method of treatment, the agent administered to a patient, such as one carrying one or more lymphoid abnormality-associated SNVs, is selected from one or more MEK / ERK inhibitors, and one or more combinations of any of the above inhibitors. In some embodiments, the agent administered is a MEK / ERK inhibitor. In some embodiments, the agent administered is one or more agents listed in Tables 1 and 2. In some embodiments, the agent administered is a MEK / ERK inhibitor and one or more agents listed in Tables 1 and 2. In some embodiments, the agent administered is a combination of 1) an mTOR inhibitor and / or a PIK3K inhibitor; and 2) one or more MEK / ERK inhibitors. In some embodiments, when the agent is an mTor inhibitor, rapamycin and / or BEZ-235 (dactolisib) are administered. In certain embodiments, the one or more mTOR inhibitors, the one or more PIK3K inhibitors, and / or the one or more MEK / ERK inhibitors have an IC50 of less than 100 μM, less than 10 μM, less than 1 μM, less than 100 nM, less than 10 nM, or less than 1 nM.

[0013] In some embodiments, the patient does not have an SNV in PTPN11. In some embodiments, the patient does not have an SNV in BRAF. In some embodiments, the patient does not have an SNV in KRAS. In some embodiments, the patient does not have an SNV in SOS1. In some embodiments, the patient does not have an SNV in ITGA9.

[0014] In some embodiments, the agents in Tables 1 and 2 are used in combination. These combinations include, but are not limited to, a) ridaforolimus and trametinib, b) ridaforolimus and selumetinib or cobimetinib, c) BEZ235 and selumetinib, d) omipalisib and selumetinib or trametinib, e) everolimus and trametinib or selumetinib, f) sirolimus, ridaforolimus and selumetinib, g) sirolimus, ridaforolimus and trametinib, h) torquinib and trametinib, i) BEZ235, torquinib and trametinib, and j) sirolimus and gedatricib and trametinib. In other embodiments, the treatment further comprises administering systemic chemotherapy, interferon alpha, radiation therapy, and / or surgery.

[0015] In some embodiments, the SNV is selected from SNVs selected from c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R (PTPN11 gene), c.35G>A:pG12D (KRAS gene), c.1403T>C:pF468S (BRAF gene), a c.2536G>A:pE846K (SOS1 gene), and c.1236+4A>G in the ITGA9 gene with a compound mutation comprising c.289T>G:p.C97G, where "c." indicates the encoded DNA sequence and "p." indicates the protein sequence.

[0016] In some embodiments, the diagnostic method includes detecting one or more of the SNVs described above. In some embodiments, the diagnostic method further includes administering to the subject one or more agents known to treat lymphatic abnormalities. In some embodiments, the agent is a MEK / ERK inhibitor. In some embodiments, the administered agent is one or more agents listed in Tables 1 and 2. In some embodiments, the administered agent is a MEK / ERK inhibitor and one or more agents listed in Tables 1 and 2. In some embodiments, the administered agent is a combination of 1) an mTOR inhibitor and / or a PIK3K inhibitor, and 2) one or more MEK / ERK inhibitors. The administration of the agent results in one or more of the following improvements: improved lymphatic structure, reduced chylous effusion, improved respiratory function, allowing for tapering of concomitant medications, and / or increased survival rate.

[0017] In some embodiments, the diagnostic method comprises detecting one or more of c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R in the PTPN11 gene and administering at least one or more MEK / ERK inhibitors, alone or in combination. In other embodiments, the agent is selected from Tables 1-2, which provides one or more of improved lymphatic vasculature, reduced bile in pleural effusion, improved respiratory function, allowing for tapering of concomitant medications, or improved survival.

[0018] In some embodiments, the diagnostic method comprises detecting c.1403T>C:pF468S in the BRAF gene. In some embodiments, the diagnostic method further comprises treating said patient with one or more MEK / ERK inhibitors, alone or in combination. In other embodiments, the agent is selected from Tables 1-2, thereby improving one or more of: improving lymphatic structure, reducing ethylation of pleural effusion, improving respiratory function, allowing for tapering of concomitant medications, or increasing survival.

[0019] In some embodiments, the diagnostic method includes detecting c.35G>A:pG12D in KRAS and administering one or more mTor inhibitors and one or more MEK / ERK inhibitors, alone or in combination. In other embodiments, at least one agent selected from Tables 1-2 is administered to improve one or more of lymphatic vasculature, reduce chylopleural effusion, improve respiratory function, allow for tapering of concomitant medications, or improve survival.

[0020] In some embodiments, the diagnostic method comprises detecting c.2536G>A:pE846K in the SOS1 gene. In some embodiments, the diagnostic method further comprises treating said patient with one or more MEK / ERK inhibitors, alone or in combination. In other embodiments, an agent from Tables 1-2 is selected that improves one or more of the following: improving lymphatic structure, reducing bile in pleural effusion, improving respiratory function, allowing for tapering of concomitant medications, or increasing survival rate.

[0021] In some embodiments, the diagnostic method comprises detecting c.1236+4A>G and / or c.289T>G:p.C97G in the ITGA9 gene and administering one or more MEK / ERK inhibitors, alone or in combination. In some embodiments, at least one agent from Tables 1-2 is administered to improve one or more of lymphatic structure, reduce pleural effusion bile, improve respiratory function, allow tapering of concomitant medications, or increase survival.

[0022] ERK / MEK inhibitors suitable for treatment include, but are not limited to, selumetinib (AZD6244), PD0325901, trametinib (GSK1120212), PD184352 (CI-1040), pimasertib (AS-703026), TAK-733, AZD8330, binimetinib (MEK162, ARRY-162, ARRY-438162), SL-327, refametinib (RDEA119, Bay 86-9766), and cobimetinib (GDC-0973, RG7420).

[0023] In some embodiments, the c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R of the PTPN11 gene, c.35G>A:pG12D of the KRAS gene, a c.1403T>C:pF468S of the BRAF gene, a c.1403T>C:pF468S of the SOS1 gene, The step of assaying the spread to determine whether single nucleotide variations (SNVs) are present at one or more of c.2536G>A:pE846K, and c.1236+4A>G and c.289T>G:p.C97 of the ITGA9 gene further comprises analyzing the polynucleotide sample to determine the presence of said SNVs by performing a process selected from the group consisting of detection of specific hybridization, measurement of allele size, restriction fragment length polymorphism analysis, allele-specific hybridization analysis, single nucleotide primer extension reaction, and sequencing of the amplified polynucleotide.

[0024] In some embodiments, the biological sample comprises DNA.

[0025] In some embodiments, the biological sample comprises RNA.

[0026] In some embodiments, the nucleic acid comprising the SNV(s) is obtained from an isolated cell of a human patient.

[0027] In some embodiments, the isolated vector encodes a nucleic acid having an SNV, the SNV being selected from a compound mutation consisting of c.1504T>G:pS502A, c.1510A>G:pM504V, and / or a c.1507G>C:pG503R in the PTPN11 gene, c.35G>A:pG12D in KRAS, c.1403T>C:pF468S in the BRAF gene, c.2536G>A:pE846K in the SOS1 gene, c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene.

[0028] In some embodiments, the host cell comprises an isolated vector encoding a nucleic acid having an SNV. In some embodiments, a transgenic animal comprises the host cell. In some embodiments, the transgenic animal is a mouse or a zebrafish.

[0029] In some embodiments, the method of screening for the effect of an agent comprises contacting a host cell or transgenic animal with one or more inhibitors described herein, alone or in combination, including agents in Tables 1-2. In some embodiments, the effect of the agent being screened is tail rescue or branch rescue in zebrafish.

[0030] In some embodiments, a method for identifying an agent that alters cell signaling comprises providing cells expressing a nucleic acid comprising at least one SNV as described above and cells expressing a cognate wild-type sequence lacking the SNV, contacting both cell populations with a test agent, and analyzing whether the agent alters cell signaling in cells harboring the nucleic acid comprising the SNV compared to cells lacking the SNV. [Brief description of the drawings]

[0031] [Figure 1] Figure 1. Overexpression of mutant BRAF and PTPN11 in Ea.hy926 cell line activates ERK signaling. [Diagram 2]Figure 2. Overexpression of BRAF F486S in the Ea.hy926 cell line alters cell morphology and actin organization. [Diagram 3] Figure 3. Treatment with the MEK inhibitor trametinib increases surface staining of VE-cadherin and filamentous actin in cells overexpressing BRAF WT and F486S mutant. [Figure 4A] Figures 4A-4B. MEK inhibitors reduce ERK activation / phosphorylation in cells overexpressing BRAFWT and F486S mutant. [Figure 4B] FIG. 4B - MEK inhibitors reduce ERK activation / phosphorylation in cells overexpressing BRAFWT and F486S mutant. [Figure 5A] Figures 5A-5B. MEK inhibitors suppress ERK activation / phosphorylation in cells overexpressing PTPN11 mutants (Figure 5A). [Figure 5B] MEK inhibitors suppressed ERK activation / phosphorylation in cells overexpressing PTPN11 mutants (Figure 5B). [Figure 6] Figure 6. Effect of PD0325901 on HDLECs expressing ARAF mutant S214P. The differences in cell morphology induced by ARAF-S214P were rescued by PD0325901, as indicated by decreased levels of pERK and increased accumulation of VE-cadherin at the cell membrane. Green is HA staining, which marks ARAF-expressing cells, and red is VE-cadherin staining. [Figure 7] Figure 7. Effect of CI1040 on HDLECs expressing ARAF mutant S214P. The differences in cell morphology induced by ARAF-S214P were restored by CI1040, as shown by the decreased levels of pERK and increased accumulation of VE-cadherin at the cell membrane. Green is HA staining, which marks ARAF-expressing cells, and red is VE-cadherin staining. [Figure 8]Figure 8. Effect of Pimasertib on HDLECs expressing ARAF mutant S214P. The differences in cell morphology induced by ARAF-S214P were rescued by Pimasertib, as shown by the decreased levels of pERK and increased accumulation of VE-cadherin at the cell membrane. Green is HA staining, which marks ARAF-expressing cells, and red is VE-cadherin staining. [Figure 9] Figure 9. Effect of TAK-733 on HDLECs expressing ARAF mutant S214P. The differences in cell morphology induced by ARAF-S214P were rescued by TAK-733, as indicated by decreased levels of pERK and increased accumulation of VE-cadherin at the cell membrane. Green is HA staining, which marks ARAF-expressing cells, and red is VE-cadherin staining. [Figure 10] Figure 10. Effect of AZD8330 on HDLECs expressing ARAF mutant S214P. The differences in cell morphology induced by ARAF-S214P were restored by AZD8330, as shown by the decreased levels of pERK and increased accumulation of VE-cadherin at the cell membrane. Green is HA staining, which marks ARAF-expressing cells, and red is VE-cadherin staining. [Figure 11] Figure 11. Effect of rifametinib on HDLECs expressing ARAF mutant S214P. The differences in cell morphology induced by ARAF-S214P were restored by rifametinib, as shown by the decreased levels of pERK and increased accumulation of VE-cadherin at the cell membrane. Green is HA staining, which marks ARAF-expressing cells, and red is VE-cadherin staining. [Figure 12] Figure 12. Effect of ulixertinib on HDLECs expressing ARAF mutant S214P. The differences in cell morphology induced by ARAF-S214P were restored by ulixertinib, as shown by the decreased phosphorylation level of the ERK substrate RSK3 and the increased accumulation of VE-cadherin at the cell membrane. Green indicates HA staining for ARAF-expressing cells, red indicates VE-cadherin staining, and white indicates DAPI staining for nuclei. [Figure 13]Figure 13. Effect of ulixertinib on HDLECs expressing BRAF mutant F486S. The difference in cell morphology induced by BRAF-F486S was restored by ulixertinib, as shown by increased VE-cadherin accumulation at the cell membrane. Green is HA staining to mark BRAF-expressing cells, red is VE-cadherin staining, and white is DAPI staining to mark nuclei. [Figure 14] Figure 14. Effect of trametinib on HDLECs expressing BRAF mutant F486S. The differences in cell morphology induced by BRAF-F486S were restored by trametinib, as shown by the decreased phosphorylation level of ERK and increased VE-cadherin accumulation at the cell membrane. Green is HA staining to mark BRAF-expressing cells, red is VE-cadherin staining, and white is DAPI staining to mark nuclei. [Figure 15] Figure 15. Effect of trametinib on HDLECs expressing the RAF1 mutant T145P. The differences in cell morphology induced by RAF1-T145P were restored by trametinib, as shown by the decreased phosphorylation level of ERK and the increased accumulation of VE-cadherin at the cell membrane. [Figure 16] Figure 16. Effects of ulixertinib and trametinib on HDLECs expressing KRAS mutant G12D. The differences in cell morphology induced by KRAS-G12D were restored by trametinib and ulixertinib, as indicated by the decreased phosphorylation level of ERK and increased accumulation of VE-cadherin at the cell membrane. [Figure 17] Figure 17. Weak activation of p-ERK induced by RIT1 mutations. [Figure 18] Figure 18. Three-dimensional lymphocyte spheroid sprouting assays show increased sprouting activity of HDLECs expressing the three EPHB4 mutations compared to EPHB4-WT, as measured by sprout length at bottom. Both rapamycin and OSI-027 were able to rescue the increased sprouts induced by the mutation L778_G779insLMGS. [Figure 19]Figure 19. Treatment with MEK inhibitors and BEZ235 significantly improved KRAS mutation G12D-induced edema. [Figure 20] Figure 20. Mosaic expression of the S502A or G503R mutation in PTPN11 resulted in a mild lymphatic vascular abnormality phenotype. [Figure 21] Figure 21. CRISPR knockout of rasa1a and rasa1b around residue 749 results in large edema formation in zebrafish, whereas single targeting (rasa1a or rasa1b) or ATG targeting does not result in a phenotype. [Fig. 22A-E] Figures 22A-22G. Clinical features and molecular analysis in the lead proband of lymphatic anomalies. Figure 22A) Coronal slice of T2-weighted non-contrast lymphangiogram showing a large pericardial effusion (arrow). Figure 22B) Maximum intensity projection of dynamic contrast-enhanced MRI lymphangiogram of a healthy control showing normal TD draining towards the left split vein. Figure 22C) Maximum intensity projection of contrast-enhanced MRI lymphangiogram of P1 showing dilated lumbar lymphatic network (arrowhead) with retrograde portal flow and dilated, tortuous TD (arrow) draining towards the left internal split vein, further supplying retrograde perfusion to the mediastinum and pericardium (box). Figure 22D) Contrast-enhanced lymphangiogram of the boxed region in c showing the dilated and tortuous distal TD and retrograde flow towards the mediastinum, pericardium and lungs via the dilated lymphatic network originating from the distal TD (arrow). FIG. 22E) Coronal maximum intensity projection of the pelvis and genitalia showing multiple dilated ducts (arrowheads) originating from bilateral inguinal lymph nodes supplying retrograde flow to the penis and scrotum (arrows). [Figure 22F] Figure 22F) Pedigree and genotype of the recurrent ARAF mutation, c.640T>C (p.S214P), identified in an unrelated family. [Figure 22G] FIG. 22G) Schematic topology of the ARAF protein. The Ser214 residue is highly conserved in vertebrates and all RAF isoforms. [Figure 23A]Figures 23A-23J. ARAF-S214P mutation increases ERK1 / 2 activity, enhances lymphangiogenesis, alters actin cytoskeleton and VE-cadherin junctions in HDLECs, and leads to thoracic duct (TD) expansion in zebrafish, which is reversed by cobimetinib. Figure 23A), transfection of HEK293T cells with ARAF mutants impairs binding to 14-3-3 proteins and increases p-ERKs. Normalized 14-3-3 / FLAG ratios are shown in the right panel, showing reduced co-immunoprecipitation of 14-3-3 proteins in the mutants. Data are shown as mean ± sem of three independent experiments. Two-tailed matched paired t-test (4 degrees of freedom), ****P=8.6×10-6. Images were cropped for better visualization. [Figure 23B] (Fig. 23B), transfection of ARAF mutants in HEK293T cells induces increased expression of p-ERK1 / 2 compared to cells expressing the WT (**P=0.0026; two-tailed unpaired t-test; df=8). Phosphorylation of AKT, p70S6K, mTOR and p38 was not altered by ARAF-S214P. Normalized ratios are shown in the boxplots on the right (all points shown, from minimum to maximum), with the center line indicating the median, the box limits the interquartile range and the whiskers indicating the data range from minimum to maximum. Six independent experiments were performed. Images were cropped for better visibility. [Fig. 23C-D]FIG. 23C) Primary HDLECs transfected with ARAF-WT or ARAF-S214P were cultured with increasing concentrations of trametinib. Results with cells from three independent transfections were quantified and graphed in a scatter plot with each individual value overlaid as a dot. Data are shown as mean ± sem (error bars) of three independent experiments. Introduction of ARAF-S214P significantly increased the levels of p-ERKs (*P=0.03; two-tailed unpaired t-test with 4 df). Trametinib treatment led to a significant decrease in p-ERKs (*P=0.02 for 100 nM trametinib treatment and 300 nM trametinib treatment; *P=0.01 for 1,000 nM trametinib treatment and 3,000 nM trametinib treatment, two-tailed unpaired t-test with 4 df); NS, not significant. Images were cropped for better visibility. Figure 23D) Three-dimensional lymphocyte spheroid sprouting assay shows increased sprouting activity in HDLECs expressing ARAF-S214P compared to ARAF-WT, as measured by both sprout number (***P=0.0002) and basal sprout length (***P=0.0005). Two-tailed t-test with 22 df. Moreover, culturing spheroids with increasing concentrations of trametinib decreased the number of sprouts at concentrations of 30 nM (****P=4.68×10-5; df=25), 100 nM (***P=9.5×10-4; df=23) and 300 nM (***P=4.4×10-4; df=24) and the length of sprouts at concentrations of 30 nM (***P=1.8×10-4; df=25), 100 nM (**P=0.001; df=23) and 300 nM (***P=3.2×10-4; df=24), respectively (two-tailed paired t-test). Three experiments performed with independent transfection of HDLECs were quantified and the points of all three experiments were plotted in an interleaved boxplot (three to six spheroids per experiment). The center line represents the median, the box limits represent the interquartile range, and the whiskers represent the range from the minimum to the maximum of the data. [Figure 23E]Fig. 23E) ARAF mutants affect VE-cadherin localization (****P=1.88×10-26) and trametinib treatment increases cell surface localization of VE-cadherin (****P=1.63×10-19). Red arrowheads indicate cell membrane staining and yellow arrowheads indicate intracellular staining. Intracellular and cell membrane staining was quantified in three independent experiments with transfected HDLECs, and the points from the three experiments are plotted in the boxplots (minimum to maximum) on the left (75 points per condition). The central line is the median, the box limits are the interquartile range, and the whiskers represent the range of data from minimum to maximum. Two-sided diagonal t-test with 148 df, NS is not significant. The maximum length and width of the cells from the experiment in Fig. 23E were measured, and the length-to-width ratio was calculated and plotted in the boxplots (minimum to maximum; bottom right) on the right. The central line represents the median, the box limits represent the interquartile range, and the whiskers represent the data range from minimum to maximum. ARAF-S214P expression significantly increased the aspect ratio (****P=9.57×10-15), and treatment with trametinib normalized the aspect ratio (****P=7.51×10-17; two-sided paired t-test with 148 df; NS, not significant). [Figure 23F] Figure 23F) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay shows that ARAF-S214P does not result in increased proliferation in transfected HDLECs. Metabolic activity is measured at two wavelengths (550 nm and 700 nm). Triplicate well contents (n=3 independent wells) were harvested at the indicated times as described in Methods. Trend lines show the average value for each transduction agent at each time point, and dots show measurements for all data points. This experiment is representative of results obtained from two independent retroviral transductions. [Fig. 23G-J]Figure 23G-I) Top: Overview of the lymphatic system in fish. The white box indicates the area investigated in Figure 23G-I, which shows an overlay (maximum intensity projection) of confocal scans. In Figure 23G-I, TDs and posterior cardinal veins (PCVs) are displayed in green (gg(mrc1a:EGFP)) and TDs are shown with dotted lines. ARAF transgenic (mrc1a:araf) cells are displayed in red (green + red → yellow). Figure 23G) Expression of ARAF-S214P leads to intense expansion of TDs. Figure 23H) Expression of ARAF-WT (red) does not affect the morphology of TDs and PCVs (both green). Figure 23I) Cobimetinib (1 uM) partially reverses the expansion induced by the mutation. Three independent experiments were repeated, with similar results to Figure 23G-I. (Fig. 23J), Phenotypic scoring categories for somite expansion with and without cobimetinib treatment: normal, moderate expansion (TDs are expanded but can be separated from PCV), and severe expansion (TDs and PCVs overlap). Cobimetinib treatment significantly inhibited severe expansion (****P=1.33×10-5; one-tailed unpaired t-test; blue histogram) and restored normal morphology (***P=0.00051; one-tailed unpaired t-test; green histogram). A total of 40 larvae and 120 somites were analyzed in three independent experiments. Data are shown as mean ± sem of three independent experiments. Raw blot images are provided as source data. [Figure 24A] Figure 24A-24F. Pulmonary function tests and clinical images of lead proband before and after MEK inhibitor treatment. Figure 24A) Pulmonary function test results before and after MEK inhibitor treatment. Note the significant improvement in all spirometry measures (marked in red): FEV1 from 23% to 42% of predicted, TLC from 29% to 56% of predicted, and maximum inspiratory pressure (MIP) from 71% to 115% of predicted. FVC is forced viability, FEF25-75 is mid-forced expiratory flow, RV is residual volume, RV / TLC is the ratio of RV to TLC, DLCO[Hb] is the pulmonary diffusion capacity for carbon monoxide corrected for hemoglobin, DLCO / VA is DLCO divided by alveolar volume (VA), MEP is maximum expiratory pressure, and O2 Sat is oxygen saturation. [Figure 24B] Figure 24B) T2-weighted non-contrast coronal maximum intensity projections of lymphatic vessels immediately prior to initiation of medical therapy (left) and 12 months after initiation of MEK inhibitor therapy (right) show nearly complete resorption of the massively dilated and beaded subcutaneous lymphatic vessels. [Figure 24C] Figure 24C) Coronal maximum intensity projection of contrast lymphangiogram of the pelvis and chest before treatment (left) shows few central lymphatics, no central lymphatic flow above the diaphragm, and profusely dilated and beaded bilateral subcutaneous ducts draining along the abdominal wall. Twelve months after the start of treatment (right), the dilated subcutaneous ducts have been absorbed and a new, more normal-appearing lymphatic network has formed extending along the abdominal wall into the chest. [Figure 24D] Figure 24D) Coronal maximum intensity projection of contrast lymphangiogram of the pelvis and thigh before treatment (left) also shows fewer vessels in the thigh and more dilated and beaded lymphatic vessels. After treatment (right), the abnormally dilated vessels have been resorbed and a new, more normal-appearing lymphatic network has formed. [Figure 24E] Figure 24E) Chest X-rays taken before treatment (left) and 12 months after treatment (right) show a decrease in pleural effusion and a significant improvement in lung capacity. [Figure 24F] Fig. 24F) Growth record of the patient (left). Treatment with trametinib was started just before the age of 13 years (*), and improvement of lymphedema and clinical symptoms was observed approximately 3-6 months after the start of treatment. The image on the top right is a photograph of the patient's lower extremities at peak weight and immediately after removing compression stockings. The image on the bottom right is a photograph of the corresponding lower extremities. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The Ras / mitogen-activated protein kinase (MAPK) pathway plays a key role in cell proliferation, migration, differentiation, and apoptosis, all of which are essential for normal development. Central duct lymphatic malformation (CCLA) is a complex lymphatic malformation characterized by lymphatic dilation, lymphatic motility disorder, and distal lymphatic obstruction. CCLA was first reported by Trenor III, Chaudry, Clemens et al., and in 2015 was classified as a channel lymphatic malformation by the International Society for the Study of Vascular Anomalies (ISSHA). Clinical manifestations include chylothorax, hylous ascites, lymphatic leakage or reflux, and limb swelling, which show a pattern that largely overlaps with the closely related diagnosis generalized lymphatic malformation (GLA). We recently identified gain-of-function mutations in ARAF as a cause of lymphatic malformations including LAM, GLA, and CCLA. Here, we introduce the gain-of-function mutations of KRAS, BIRAF, and PNPN11 as causative mutations of lymphoid abnormalities including LAM, GLA, and CCLA. We also revealed that mutations in SOS1, ITAG9, RASA1, RAF1, RIT1, PIEZO1, EPHB4, NF1, CBL, and ARAF contribute to the onset of this disease.

[0033] Lymphatic disorders are a rare and devastating disease spectrum with mostly unknown causes, and treatment varies depending on the patient's symptoms. Identifying the causative genes could allow the development of affordable treatments in line with precision medicine practices.

[0034] In Example II, we characterized a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with a progressive abnormal lymphatic disease unresponsive to conventional sirolimus therapy and in another unrelated adult patient. The mutation resulted in the loss of a conserved phosphorylation site. Cells transfected with ARAF-S214P showed increased ERK1 / 2 activity, enhanced lymphatic vessel formation, and disruption of the actin cytoskeleton and VE-cadherin junctions, which could be restored with the MEK inhibitor trametinib. The functional relevance of this mutation was also verified by recapitulating the lymphatic phenotype in a zebrafish model and rescuing the abnormal phenotype with a MEK inhibitor. Subsequent treatment with a MEK inhibitor resulted in dramatic clinical improvement in the patient, including remodeling of the lymphatic system and resolution of lymphedema, marked improvement in pulmonary function tests, cessation of supplemental oxygen, and near normalization of daily life. Our results are a prime example of how genetic classification and mechanistic understanding can guide biologically based treatment, which in our case was life-saving.

[0035] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the invention as defined by the appended claims.

[0036] Before describing the teachings of the present invention in detail, it is to be understood that the present disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates, reference to "a cell" includes a plurality of cells, and so forth.

[0037] It will be understood that there is an implicit "about" before temperatures, concentrations, times, etc. discussed in this disclosure such that minor and insubstantial deviations are within the scope of the teachings herein. Also, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that both the foregoing general and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0038] Unless otherwise noted in the specification above, embodiments herein that are described as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments herein that are described as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components. Also, herein, embodiments that are described as "consisting essentially of" various components are also considered to be "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0039] The section headings used herein are for organizational purposes and are not to be construed as limiting the desired subject matter in any manner. In the event that a document incorporated by reference conflicts with any term defined herein, the present specification controls. While the teachings of the present invention are described in conjunction with various embodiments, it is not intended that the teachings of the present invention be limited to such embodiments. On the contrary, the teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0040] Additionally, a compound "selected from the group consisting of" refers to one or more compounds in the list below, including mixtures (i.e., combinations) of two or more compounds. According to the present invention, an isolated or biologically pure molecule is a compound that has been removed from its natural environment. Thus, "isolated" and "biologically pure" do not necessarily reflect the extent to which a compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthesis techniques, or can be produced by any such chemical synthesis route.

[0041] "Lymphatic abnormalities" refers to diseases or disorders characterized by abnormal formation of lymphatic vessels and overgrowth of tissue. Non-limiting examples of lymphatic abnormalities include "lymphangiomatosis" or "lymphangiectasia" (collectively referred to herein as LAM), lymphangioma, generalized lymphatic malformation (GLA), and chyle exudate, generalized lymphangioma. Generalized cystic hemangiomatosis, multiple lymphangiectasia, generalized lymphatic malformation, CCLA, diffuse lymphatic malformation, Kaposiform LAM, and Gorham-Stout disease (GSD), a rare vascular disorder of lymphatic origin characterized by progressive osteolysis.

[0042] Clinically, lymphangiomas are classified into several types. These include (1) simplex, which is composed of thin-walled lymphatic vessels the size of capillaries. This type usually affects the skin (perilangiomas). (2) cystic lymphangiomas (or cystic hygromas): This can range in size from a few millimeters to a few centimeters, found at a young age and typically in the neck or axilla. (3) cavernous: This type is composed of dilated lymphatic vessels, often with a fibrous adventitia. This is the type that usually affects the organs of the chest, abdomen, and bones. Each of these lymphangiomas is included in the present invention.

[0043] A "single nucleotide polymorphism (SNV)" is a single base that is different from the normal base at a certain position in genomic DNA. SNVs are similar to SNPs, but SNPs generally occur with some frequency (e.g., occur in a certain percentage of the population), whereas SNVs do not have frequency information. There are millions of SNVs cataloged in the human genome. Some SNVs cause disease, while others are normal variations in the genome.

[0044] A "lymphatic abnormality-associated SNV or -specific marker" is an SNV that is associated with an increased risk of developing a lymphatic abnormality and is not found in patients without the disease. Such markers include, but are not limited to, nucleic acids, proteins encoded thereby, or other small molecules.

[0045] As used herein, the term "genetic modification" refers to a change from a wild-type or reference sequence of one or more nucleic acid molecules. Genetic modifications include, but are not limited to, SNVs and SNPs, copy number variations (CNVs), base pair substitutions, additions, and deletions of at least one nucleotide from a nucleic acid molecule of known sequence.

[0046] "Linkage" refers to the tendency of genes, alleles, loci, or genetic markers to be inherited together due to their location on the same chromosome, as measured by the recombination rate (also called the recombination rate, or θ) between two genes, alleles, loci, or genetic markers. The closer physically two loci are on a chromosome, the lower the recombination rate. Usually, when testing for association between a polymorphic site in a disease-causing gene and a disease, the recombination rate is zero, indicating that the disease and the disease-causing gene are always tailor-made. In rare cases, when a gene occupies a very large portion of the genome, recombination can be observed between a polymorphic site at one end of the gene and a causative mutation at the other end. However, when the causative mutation is the polymorphism being tested for association with the disease, no recombination is observed.

[0047] A "centimorgan" is a unit of genetic distance that denotes the linkage between two genetic markers, alleles, genes, or loci, and corresponds to a 1% probability of recombination between the two markers or loci in any meiotic event.

[0048] "Linkage disequilibrium" or "allelic association" means the preferential association of a particular allele, locus, gene or genetic marker with a particular allele, locus, gene or genetic marker at a nearby chromosomal location at a frequency greater than would be expected by chance for the frequency of the particular allele in a population.

[0049] As used herein, "solid matrix" refers to any format, including beads, microparticles, microarrays, surfaces of microtiter wells or test tubes, dipsticks, filters, etc. The matrix material may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran, or agarose. The solid matrix may include nucleic acids immobilized thereon such that they are not removed from the matrix in solution.

[0050] "Target nucleic acid" as used herein refers to a previously defined region of nucleic acid present in a complex nucleic acid mixture, the defined wild type region being one that contains at least one known nucleotide mutation that may or may not be associated with lymphoid abnormality. Nucleic acid molecules may be isolated from natural sources by cDNA cloning or subtractive hybridization, or may be synthesized manually. Nucleic acid molecules may be synthesized manually by triester synthesis or using an automated DNA synthesizer.

[0051] As used herein with respect to nucleic acids, the term "isolated nucleic acid" as applied to DNA means a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. For example, an "isolated nucleic acid" may consist of a DNA molecule inserted into a vector, such as a plasmid or viral vector, or integrated into the genomic DNA of a prokaryote or eukaryote. An "isolated nucleic acid molecule" may also include a cDNA molecule. An isolated nucleic acid molecule inserted into a vector may also be referred to herein as a recombinant nucleic acid molecule.

[0052] With respect to RNA molecules, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. The term may also refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues) and exists in "substantially pure" form.

[0053] The use of the term "enriched" with respect to nucleic acids means that the particular DNA or RNA sequence constitutes a significantly higher proportion (2-5 fold) of the total DNA or RNA present in the cells or solution of interest compared to normal cells or the cells from which the sequence was taken. This may be caused by a preferential decrease in the amount of other DNA or RNA present, by a preferential increase in the amount of the particular DNA or RNA sequence, or by a combination of the two. However, it should be noted that "enriched" does not mean that other DNA or RNA sequences are absent, only that the relative amount of the sequence of interest has been significantly increased.

[0054] Also, for some purposes, it is advantageous for the nucleotide sequence to be in a purified form. The term "purified" with respect to nucleic acids does not require absolute purity (such as a homogeneous preparation), but instead indicates that the sequence is relatively purer than its natural environment.

[0055] The term "complementary" refers to two nucleotides that can form multiple favorable interactions with each other. For example, adenine is complementary to thymine and can form two hydrogen bonds. Similarly, guanine and cytosine are complementary because they can form three hydrogen bonds. Thus, if a nucleic acid sequence contains the base sequence thymine, adenine, guanine, cytosine, then the "complement" of this nucleic acid molecule would be a molecule that contains adenine in place of thymine, thymine in place of adenine, cytosine in place of guanine, and guanine in place of cytosine. A complement can contain a nucleic acid sequence that forms an optimal interaction with the parent nucleic acid molecule, such that the complement can bind to the parent molecule with high affinity.

[0056] With respect to single-stranded nucleic acids, particularly oligonucleotides, the term "specifically hybridize" refers to the association between two single-stranded nucleotide molecules having sufficiently complementary sequences to allow such hybridization under predetermined conditions commonly used in the art (sometimes referred to as "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, substantially excluding hybridization of the oligonucleotide with a single-stranded nucleic acid of a non-complementary sequence. For example, specific hybridization can refer to a sequence that hybridizes to any lymphoid abnormality specific marker nucleic acid, but not to other nucleotides. Such markers include, for example, lymphoid abnormality specific markers shown in the tables contained herein. Appropriate conditions that allow specific hybridization of single-stranded nucleic acid molecules of differing complementarity are well known in the art.

[0057] For example, one common formula for calculating the stringency conditions necessary to achieve hybridization between nucleic acid molecules with specific sequence homology is given below (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989)): T m =81.5℃+16.6Log[Na+]+0.41(% G+C)-0.63(% formamide)-600 / #bp with double structure As an example of the above formula, if [Na+] = [0.368], 50% formamide is used, the GC content is 42%, and the average probe size is 200 bases, then T m The T of the DNA double strand is 57°C. mdecreases by 1-1.5°C for every 1% decrease in homology. Thus, targets with approximately 75% or more sequence identity would be observed using a hybridization temperature of 42°C. The stringency of the hybridization and washing depends primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe and target, hybridization is usually performed at a calculated T m Washing conditions should be as stringent as possible, depending on the degree of identity of the probe to the target. In general, washing conditions should be chosen based on the T m The temperature is selected to be about 12-20°C lower than the temperature at which the nucleic acid is hybridized. In a particular aspect, for the nucleic acid of the present invention, moderate stringency hybridization is defined as hybridization at 42°C with 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg / ml denatured salmon sperm DNA, followed by washing at 55°C for 15 minutes with 2X SSC and 0.5% SDS. High stringency hybridization is hybridization at 42°C with 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg / ml denatured salmon sperm DNA, followed by washing at 65°C for 15 minutes with 1X SSC and 0.5% SDS. Very high stringency hybridization was performed using 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg / ml denatured salmon sperm DNA at 42°C, followed by a 15-minute wash in 0.1X SSC and 0.5% SDS at 65°C.

[0058] The term "oligonucleotide" as used herein is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably three or more ribo- or deoxyribonucleotides. The exact size of the oligonucleotide depends on various factors and the particular application and method of use of the oligonucleotide. Oligonucleotides, including probes and primers, can be any length from 3 nucleotides to the full length of the nucleic acid molecule, and expressly include any possible number of contiguous nucleic acids from 3 to the full length of the polynucleotide. Preferably, the oligonucleotide is at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, and more preferably at least about 20 nucleotides in length.

[0059] As used herein, a "probe" refers to an RNA or DNA oligonucleotide, polynucleotide, or nucleic acid, whether naturally occurring, such as a purified restriction enzyme digest, or synthetic, that is capable of annealing or specifically hybridizing to a nucleic acid having a sequence complementary to the probe. The probe may be single-stranded or double-stranded. The exact length of the probe will depend on many factors, including temperature, source of probe, and use of the method. For example, in diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe (in certain cases, a nucleic acid associated with a designated rs number associated with a single nucleotide polymorphism available in the dbSNP database) typically contains 15-25, 15-35, 20-50, or 100 or more nucleotides, but may contain fewer nucleotides if the site of the SNV is included in the probe. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. That is, the probes must have sufficient complementarity to be able to "specifically hybridize" or anneal to their respective target strands under a predefined set of conditions. Thus, the sequence of the probe does not have to reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, the probe may be interspersed with non-complementary bases or longer sequences, as long as the probe sequence is sufficiently complementary to the sequence of the target nucleic acid to anneal specifically to it.

[0060] "Primer" refers to a single- or double-stranded oligonucleotide of RNA or DNA, derived from a biological system, produced by restriction enzyme digestion, or synthesized, that, when placed in the appropriate environment, can function as an initiator of template-dependent nucleic acid synthesis. Given a suitable nucleic acid template, a suitable nucleic acid nucleoside triphosphate precursor, a polymerase enzyme, suitable coenzymes, and suitable conditions such as temperature and pH, the primer can be extended at its 3' end by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. Primers may vary in length depending on the specific conditions and requirements of the application. For example, in diagnostic applications, oligonucleotide primers are typically 1525, 15-40, 20-50, etc., or more nucleotides in length. A primer must have sufficient complementarity to the desired template to facilitate synthesis of the desired extension product. That is, it must be capable of annealing to the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in proper juxtaposition for use in initiating synthesis by a polymerase or similar enzyme. It is not essential that the sequence of the primer exhibits an exact complementarity to the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5' end of a complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to provide a functional template-primer complex for synthesis of an extension product.

[0061] Polymerase chain reaction (PCR) is described in US Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the disclosures of which are incorporated herein by reference in their entireties.

[0062] "siRNA" refers to a molecule involved in the RNA interference process for sequence-specific post-transcriptional gene silencing or gene knockdown by providing small interfering RNA (siRNA) with sequence homology to the target gene. Small interfering RNA (siRNA) is synthesized in vitro or generated by cleavage from long dsRNA by RNase III to mediate sequence-specific mRNA degradation. Preferably, the siRNA of the present invention is chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. siRNA can be synthesized as two separate complementary RNA molecules or as a single RNA molecule with two complementary regions. Commercial sources of synthetic RNA molecules or synthesis reagents include Applied Biosystems (Foster City, CA, USA), Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Stirling, VA, USA), ChemGenes (Ashland, MA, USA), Cruachem (Glasgow, UK), etc. Specific siRNA constructs for inhibiting Limphann angiomatosis mRNA can be, for example, 15-35 nucleotides in length, more typically about 21 nucleotides in length.

[0063] A "vector" refers to a single-stranded or double-stranded circular nucleic acid molecule that can infect, transfect, or transform a cell and replicate independently or within the genome of a host cell. A circular double-stranded nucleic acid molecule can be cut and thereby linearized by treatment with a restriction enzyme. Knowledge of vectors, restriction enzymes, and the nucleotide sequences targeted by the restriction enzymes are readily available to the skilled artisan and includes any replicon, such as a plasmid, cosmid, bacmid, phage, or virus, to which another genetic sequence or element (either DNA or RNA) can be attached and which can result in replication of the attached sequence or element. The nucleic acid molecule of the present invention can be inserted into a vector by cutting the vector with a restriction enzyme and ligating the two pieces. When cloning a gene region that contains a duplication or deletion, the skilled artisan will be well aware that flanking sequences upstream and downstream of the affected region of appropriate length (e.g., between 50-100 or more nucleotides) will be employed in the cloning process. Such vectors are useful, for example, in cell lines to study the effect of such changes on the encoded protein.

[0064] Many techniques are available to those skilled in the art to facilitate transformation, transfection, or transduction of expression constructs into prokaryotes or eukaryotes. The terms "transformation," "transfection," and "introduction" refer to methods of inserting nucleic acids and / or expression constructs into cells or host organisms. These methods include treating cells with high concentrations of salt, electric fields, detergents, etc. to make the outer membrane or wall of the host cell permeable to the nucleic acid molecule of interest, microinjection, PEG fusion, and various other techniques.

[0065] A "promoter element" is a nucleotide sequence incorporated into a vector that, once inside an appropriate cell, facilitates the binding of transcription factors and polymerases and subsequent transcription of a portion of the vector DNA into mRNA. In one embodiment, a promoter element of the invention precedes the 5' end of a lymphoid abnormality specific marker nucleic acid molecule, which is then transcribed into mRNA. The host cell machinery then translates the mRNA into a polypeptide. The promoter element can constitutively or inducibly express a coding region of interest.

[0066] Those skilled in the art will recognize that a nucleic acid vector can contain nucleic acid elements other than a promoter element and a nucleic acid encoding a lymphoid abnormality-specific marker. These other nucleic acid elements include, but are not limited to, origins of replication, ribosome binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolizing enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for purification of the polypeptide.

[0067] A "replicon" is any genetic element capable of largely self-controlled replication, e.g., a plasmid, cosmid, bacmid, plastid, phage, virus, etc. Replicons can be either RNA or DNA, and either single-stranded or double-stranded.

[0068] "Expression operon" refers to a nucleic acid segment that contains transcriptional and translational control sequences, such as promoters, enhancers, translation initiation signals (e.g., ATG and AUG codons), polyadenylation signals, and terminators, and that may facilitate the expression of a polypeptide coding sequence in a host cell or organism.

[0069] As used herein, the terms "reporter", "reporter system", "reporter gene" and "reporter gene product" refer to an operable genetic system in which a nucleic acid comprises a gene encoding a product that upon expression produces a reporter signal that is readily measurable, for example, by biological assay, immunoassay, radioimmunoassay, or colorimetric, fluorescent, chemiluminescent or other methods. The nucleic acid may be RNA or DNA, linear or circular, single-stranded or double-stranded, of antisense or sense polarity, and is operably linked to control elements required for expression of the reporter gene product. The necessary control elements will vary depending on the nature of the reporter system and whether the reporter gene is in DNA or RNA form, but may include, but are not limited to, promoters, enhancers, translation control sequences, poly A addition signals, transcription termination signals, and other elements.

[0070] The introduced nucleic acid may or may not be integrated (covalently linked) into the nucleic acid of the recipient cell or organism. For example, in bacterial, yeast, plant, and mammalian cells, the introduced nucleic acid may be maintained as an independent replicon, such as an episomal element or a plasmid. Alternatively, the introduced nucleic acid may be integrated into the nucleic acid of the recipient cell or organism, stably maintained in that cell or organism, and further inherited or inherited by cells or organisms progeny of the recipient cell or organism. Finally, the introduced nucleic acid may be present in the recipient cell or host organism only transiently.

[0071] A "selectable marker gene" is a gene whose expression confers a selectable phenotype, such as antibiotic resistance, on transformed cells.

[0072] "Operably linked" means that control sequences required for expression of a coding sequence are positioned within the DNA molecule in the appropriate position relative to the coding sequence to effect expression of the coding sequence. This same definition may also be applied to the arrangement of transcription units and other transcription control elements (e.g., enhancers) in an expression vector.

[0073] "Recombinant organism" or "transgenic organism" refers to an organism that contains a new combination of genes or nucleic acid molecules. New combinations of genes or nucleic acid molecules can be introduced into an organism using a wide range of nucleic acid manipulation techniques available to those skilled in the art. An "organism" is any living organism that is composed of at least one cell. Organisms can be as simple as a single eukaryotic cell or as complex as a mammal. Thus, the term "recombinant organism" includes recombinant cells as well as eukaryotic and prokaryotic organisms. Examples of transgenic organisms include zebrafish and mice.

[0074] The term "isolated protein" or "isolated and purified protein" may be used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the present invention. Alternatively, this term may refer to a protein sufficiently separated from other proteins with which it would be naturally associated, such that it exists in "substantially pure" form. "Isolated" does not mean to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the basic activity, such as may be present due to incomplete purification, addition of stabilizers, or incorporation into, for example, an immunogenic or pharma- ceutical acceptable formulation.

[0075] A "specific binding pair" consists of a specific binding member (sbm) and a binding partner (bp) that have a particular specificity for each other and bind preferentially to other molecules under normal conditions. Examples of specific binding pairs include antigens and antibodies, ligands and receptors, complementary nucleotide sequences, etc. Many other examples will be known to those skilled in the art. Furthermore, the term "specific binding pair" also applies when either or both of the specific binding member and the binding partner form part of a larger molecule. In embodiments where the specific binding pair consists of nucleic acid sequences, they will be of a length such that they hybridize to each other under the conditions of the assay, preferably 10 nucleotides or more in length, more preferably 15 or 20 nucleotides or more in length.

[0076] "Sample" or "patient sample" or "biological sample" generally refers to a sample that can be tested for a particular molecule, preferably a lymphatic abnormality specific marker molecule, such as the markers set forth in the table provided below. Samples include, but are not limited to, cells, bodily fluids such as blood, serum, plasma, urine, lymph, saliva, tears, pleural fluid, etc.

[0077] "Genotypic sequence information" generally refers to any information related to the sequence of a subject's DNA or RNA. Genotypic sequence information includes whole genome, whole exome, exome sequencing, or targeted sequencing of regions of interest within a subject's genome. Genotypic sequence information also includes generating data regarding the presence or absence of specific SNVs, such as those associated with lymphatic abnormalities herein. Furthermore, genotypic sequence information may include the use of probes to detect the presence and / or expression of one or more SNVs associated with lymphatic abnormalities. Examples of how probes may be used to obtain genotypic sequence information include, but are not limited to, the following: (1) in situ hybridization, (2) Southern hybridization, (3) Northern hybridization, and (4) various amplification reactions, such as polymerase chain reaction (PCR).

[0078] As used herein, the terms "agent" and "test compound" are used interchangeably to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, and animal (especially mammalian) cells and tissues. Biological macromolecules include siRNAs, shRNAs, antisense oligonucleotides, peptides, peptide / DNA complexes, and any nucleic acid-based molecule that exhibits the ability to modulate the activity of an SNV-containing nucleic acid or its encoded protein as described herein. Exemplary agents include, but are not limited to, at least one MEK inhibitor. Additional agents also include those listed in Tables 1-2. Agents are evaluated for potential biological activity by inclusion in the screening assays described herein below.

[0079] "Treatment," as used herein, covers any administration or application of a therapeutic agent to a disease in a mammal, including a human, including inhibiting the disease or its progression, suppressing or slowing the disease or its progression, arresting its occurrence, partially or completely alleviating the disease, preventing the onset of the disease, or preventing the recurrence of symptoms of the disease. Exemplary treatment methods include administering at least one MEK inhibitor, or at least one of the agents set forth in Tables 1-2, in an effective dose.

[0080] The term "inhibit" or "suppress" refers to the reduction or cessation of any event (such as ligand binding of a protein), or the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the incidence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. The suppression or reduction need not be complete. For example, in one embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or more. In another embodiment, "reduce" or "suppress" refers to the ability to cause an overall reduction of 50% or more. In yet another embodiment, "reduce" or "suppress" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0081] "Inhibitor" refers to an agent that slows or prevents a particular chemical reaction, signaling pathway, or other process, or that reduces the activity of a particular reactant, catalyst, or enzyme.

[0082] The terms "patient" and "subject" are used interchangeably to mean a mammal, including a human.

[0083] "MEK" refers to the MAPK / ERK pathway (also called the Ras-Raf-MEK-ERK pathway), which is a chain of intracellular proteins that transmit signals from receptors on the cell surface to DNA in the cell nucleus. A signal begins when a signal molecule binds to a receptor on the cell surface and ends when DNA in the nucleus expresses a protein that causes some change in the cell, such as cell division. This pathway includes many proteins, including MAPKs (mitogen-activated protein kinases, originally called ERKs (extracellular signal-regulated kinases)), which transmit signals by adding phosphate groups to adjacent proteins, acting as "on" or "off" switches.

[0084] "MEK inhibitors" or "MEK / ERK inhibitors" refer to agents that inhibit the mitogen-activated protein kinase enzymes MEK1, MEK2, and / or ERK. These can be used to affect the MAPK / ERK pathway, which is often overactivated in some cancers. The term "intracellular signaling" would include not only mTOR signaling, but other signaling pathway processes that govern cellular homeostasis or activity.

[0085] Diagnosing patients with lymphatic system disorders In some embodiments, a patient with a lymphatic abnormality is diagnosed based on the presence of SNVs after obtaining genotype sequence information from a biological sample obtained from the patient. In some embodiments, a patient with a lymphatic abnormality has one or more SNVs in a gene selected from PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, and CBL, or SNVs in linkage disequilibrium with an SNV in a gene selected from PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, and CBL associated with lymphatic abnormality. In some embodiments, the one or more SNVs are c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R in the PTPN11 gene, a c.35G>A:pG12D in KRAS, a c.1403T>C:pF468S in the BRAF gene, ac.2536G>A:pE846K in the SOS1 gene, a compound mutation consisting of c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene, or any of the mutations listed in Table 3.

[0086] In some embodiments, a report may be generated from the experimental data identifying SNVs present in a particular subject. In some embodiments, a report may be generated that identifies treatment recommendations for lymphatic abnormalities based on data on the SNV(s) identified using the genotype sequence information.

[0087] In some embodiments, the presence of one or more SNVs in a gene selected from PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, and CBL, or SNVs that are in linkage disequilibrium with an SNV in a gene selected from PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, CBL, guides treatment choices for a patient after obtaining genotype sequence information from a biological sample obtained from the patient. In some embodiments, the presence of one or more SNVs in genes selected from PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, and CBL, or SNVs that are in linkage disequilibrium with SNVs in genes selected from PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, and CBL, does not guide or influence treatment choices for a patient after genotype sequence information is obtained from a biological sample obtained from the patient.

[0088] In some embodiments, the diagnosis of lymphatic system abnormalities is made based solely on clinical symptoms, scan results, and / or family history. In some embodiments, the diagnosis of lymphatic system abnormalities is made without testing for genetic sequence information. In some embodiments, the diagnosis of lymphatic system abnormalities is made based on clinical symptoms in conjunction with genetic sequence information.

[0089] The lymphatic abnormality-associated SNVs disclosed in the present invention can be used in a variety of ways to diagnose lymphatic abnormalities.

[0090] For example, a nucleic acid containing a lymphatic abnormality-associated SNV can be used as a probe to detect the presence and / or expression of a lymphatic abnormality-specific marker. Methods in which a lymphatic abnormality-associated marker nucleic acid can be used as a probe for such an assay include, but are not limited to, the following: (1) in situ hybridization, (2) Southern hybridization, (3) Northern hybridization, and (4) assorted amplification reactions such as polymerase chain reaction (PCR).

[0091] Furthermore, assays to detect SNVs associated with lymphatic abnormalities, or the proteins encoded thereby, can be performed on any type of biological sample, including body fluids (blood, urine, serum, gastric washings, etc.), any type of cell (brain cells, white blood cells, mononuclear cells, etc.), and body tissues.

[0092] Lymphatic abnormality-associated SNV-containing nucleic acids, vectors expressing the same, lymphatic abnormality-associated SNV-containing marker proteins, and anti-lymphatic abnormality-specific marker antibodies can be used to detect lymphatic abnormality-associated SNVs in body tissues, cells, or body fluids, and to alter the expression of lymphatic abnormality-associated SNV-containing marker proteins for the purpose of detecting and diagnosing lymphatic abnormalities.

[0093] The method for detecting and / or diagnosing lymphatic abnormality based on lymphatic abnormality-associated SNV is included.The method may include detecting lymphatic abnormality-associated SNV, and the nucleic acid containing lymphatic abnormality-associated SNV in a sample is first amplified, for example by PCR, to increase the amount of template compared with other sequences present in the sample.This allows the target sequence to be detected with high sensitivity if it exists in the sample.This initial step can be avoided by using highly sensitive array technology, which is gaining importance in the art.

[0094] On the other hand, new detection techniques overcome this limitation and allow the analysis of small samples containing as little as 1 μg of total RNA. Unlike traditional fluorescence techniques, the Resonant Light Scattering (RLS) technique allows the detection of small amounts of mRNA with multiple reads using a biotin-labeled hybridized target and an anti-biotin antibody. Also, as an alternative to PCR amplification, the Planar Waveguide Technology (PWG) technique improves the signal-to-noise ratio and reduces background interference. Both techniques are commercially available from Qiagen Inc. (USA).

[0095] Thus, any of the techniques described above can be used to detect or quantify the expression of SNV markers associated with lymphatic abnormalities and accordingly diagnose lymphatic abnormalities or the risk of developing such abnormalities.

[0096] Care for patients with lymphatic system disorders The elucidation of the role that the lymphatic abnormality-associated SNVs described herein play in modulating the lymphatic abnormality phenotype will facilitate the repurposing of existing therapies useful for treating lymphatic abnormalities and the development of new therapies. In some embodiments, the invention comprises administering one or more mTOR inhibitors, one or more PIK3K inhibitors, and / or one or more MEK inhibitors (e.g., one or more agents in Tables 1-2) to a patient having a lymphatic abnormality.

[0097] In some embodiments, patients with lymphatic abnormalities to be treated are diagnosed based on symptoms and a positive family history of lymphatic abnormalities. In some embodiments, various scanning techniques, such as plain film radiography, bone scans, computed tomography, magnetic resonance imaging, and lymphoscintigraphy, are used along with clinical symptoms to diagnose lymphatic abnormalities. In some embodiments, a biopsy is performed to diagnose lymphatic abnormalities. In some embodiments, lymphatic abnormalities are diagnosed based on lymphatic vessel overgrowth. In some embodiments, lymphatic abnormalities are diagnosed based on abnormal formation of lymphatic vessels. In some embodiments, lymphatic abnormalities are diagnosed based on chylous effusions, including pericardial, pleural, or peritoneal fluids.

[0098] In some embodiments, the patient having a lymphatic abnormality to be treated is diagnosed according to the diagnostic methods described herein.

[0099] In some embodiments, one or more MEK inhibitors (e.g., one or more agents of Tables 1-2; Example II) are useful in the preparation of a medicament for treating lymphatic abnormalities. One or more agents may be formulated with pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials familiar to those of skill in the art. Desirably, such materials are non-toxic and do not inhibit the efficacy of the active ingredient. The exact nature of the carrier or other materials may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, intranasal, aerosolized, intramuscular, and intraperitoneal routes. In vitro systems or transgenic organisms containing mutations associated with lymphatic abnormalities may be used to select specific agents for human treatment.

[0100] Agents useful for treatment include, but are not limited to, those in Table 1 and Table 2. Some agents may be listed in both Table 1 and Table 2, and there is no significance in not listing an agent in both tables. [Table 1] TIFF2025020109000002.tif152141TIFF2025020109000003.tif171141TIFF2025020109000004.tif122141

[0101] Table 2 provides agents that can be used alone or in combination with any of the agents of Table 1 or Table 2 to treat lymphatic disorders. [Table 2] TIFF2025020109000006.tif106141 [ka]

[0102] To treat individuals with lymphatic abnormalities or to alleviate the signs and symptoms of disease, appropriate drugs that target the genes disclosed herein can be administered in combination to provide therapeutic benefit to the patient. Such drugs should be administered in effective doses.

[0103] Once the genetic alterations are identified, treatments are devised to modulate the biological and signaling pathways affected by the gene. For example, if it is desired to inhibit the MAPK (MEK1 / MEK2) and ERK pathways, MEK inhibitors can be used alone or in combination with other MEK / ERK inhibitors. In certain embodiments, treatment involves administration of an agent listed in Tables 1 or 2, such as an mTOR inhibitor, together with a PIK3K inhibitor. In other embodiments, an mTOR inhibitor and a MEK / ERK inhibitor are combined to provide a therapeutic benefit to the patient. In another approach, a PIK3K inhibitor is combined with a MEK / ERK inhibitor to ameliorate the symptoms of the disease. For certain ARAF gain-of-function mutations described herein, an effective treatment consists of administration of a MEK / ERK inhibitor. The combinatorial therapy described above can act additively. In other embodiments, the combined agents act synergistically to alleviate symptoms.

[0104] First, a biological sample and genotype information are obtained from the patient. The genetic information obtained from the nucleic acid contained in the sample is then evaluated for the presence or absence of SNVs associated with lymphatic abnormalities, for example. The presence of these mutations, which indicate the risk of lymphatic abnormalities or the presence of disease, along with the simultaneous identification of the affected genes, provides the clinician with guidance as to which therapeutic agent is appropriate. The total therapeutic amount or dose (if two or more targets are modulated) can be administered to the subject as a single dose, or can be administered using a fractionated treatment protocol, in which multiple / separate doses are administered over a longer period of time, for example, over a daily period so that a daily dose can be administered, or over a longer period of time to administer for the desired period of time. Those skilled in the art will know that the amount of lymphatic abnormality agent required to obtain an effective dose in a subject will depend on many factors, such as the age, weight, and general health of the subject, as well as the route of administration and the number of treatments administered. Taking these factors into account, those skilled in the art will adjust the specific dose to obtain an effective dose for treating an individual with lymphatic abnormalities.

[0105] The effective amount of the lymphatic disorder treatment agent depends on the method of administration and the body weight of the individual being treated. The dosages described herein are generally for an average adult, but can be adjusted for the treatment of children. Dosages will generally range from about 0.001 mg to about 1000 mg.

[0106] In particular, in individuals suffering from more severe forms of lymphatic dysfunction, administration of lymphatic dysfunction therapeutic agents is particularly useful, for example, when administered in combination with conventional drugs for treating such diseases. Those skilled in the art will administer lymphatic dysfunction therapeutic agents alone or in combination and monitor the effectiveness of such treatment using routine methods, such as measurements of lung, intestinal, thyroid, or inflammatory function, radiological or immunological assays, or, if necessary, histopathological methods. Other drugs for the treatment of lymphatic dysfunction include systemic chemotherapy, interferon alpha therapy, radiotherapy, or surgery to alleviate the underlying symptoms of the disease.

[0107] The pharmaceutical preparation is preferably administered in an "effective amount", which is an amount sufficient to show benefit to an individual. This amount prevents, alleviates, relieves, or otherwise reduces the severity of symptoms of lymphatic system abnormalities in a patient. Treatment of a patient with lymphatic system abnormalities with an effective amount of a MEK inhibitor and / or a drug from Tables 1-2) may result in improved lymphatic structure, reduced pleural effusion, improved respiratory function, tapered use of concomitant medications, or improved survival.

[0108] The product is formulated in dosage unit form for ease of administration and uniformity of dosage. A dosage unit is a physically separate unit of pharmaceutical preparation appropriate for the patient to be treated. Each dosage should contain an amount of active ingredient calculated to produce the desired effect in combination with the selected pharmaceutical carrier. The procedure for determining the appropriate dosage unit is well known to those skilled in the art.

[0109] Dosage units can be proportionally increased or decreased depending on the patient's body weight. Appropriate concentrations for alleviating a particular medical condition can be determined by dose-concentration curve calculations, as known in the art.

[0110] Pharmaceutical compositions useful in the methods of the invention can be administered systemically in parenteral, oral solid and liquid formulations, subcutaneously, intradermally, intramuscularly, sublingually, topically, OTIC, buccal, conjunctival, dermal, dental, via electroosmosis, via cervical, sinus or trachea, enterally, epidurally, infiltratively, interstitially, intraperitoneally, intraarterially, intraarticularly, intrabiliary, intratracheal, intracardiac, intratracheal, intrasternal, intrathoracic, intracerebral, intradermal, intralymphatic, intracardiac, intraperitoneal, intranasal, transdermal, respiratory, ophthalmic, suppository, aerosol, topically, or via other known routes of administration. In addition to the agent useful for treating lymphatic abnormalities, the pharmaceutical composition may include pharma- ceutical acceptable carriers and other ingredients known to enhance and facilitate administration of the agent. Thus, such compositions can optionally include other ingredients, such as adjuvants, such as aqueous suspensions of aluminum hydroxide and magnesium hydroxide, and / or other pharma- ceutical acceptable carriers, such as saline. Other possible formulations such as nanoparticles, liposomes, resealed red blood cells, and immunologically based systems can also be used to deliver / administer the appropriate drug to a patient according to the methods of the present invention. The use of nanoparticles to deliver such drugs, and cell membrane permeable peptide carriers that can be used, are described in Crombez et al., Biochemical Society Transactions v35:p44 (2007).

[0111] Administration of an agent useful for treating a lymphatic abnormality can be performed after successfully detecting or quantifying the expression of an SNV marker associated with a lymphatic abnormality, thereby diagnosing a lymphatic abnormality or the risk of developing the same. Detecting or quantifying the expression of an SNV marker associated with a lymphatic abnormality can select a particular agent for use in treatment. Detecting or quantifying the expression of an SNV marker associated with a lymphatic abnormality can indicate that a particular treatment is not appropriate for a subject.

[0112] In other embodiments, treatment for lymphatic abnormalities may be based on a clinical diagnosis of disease, and treatment may be initiated without detection or quantification of gene sequence information.In other embodiments, treatment for lymphatic abnormalities may be based on a clinical diagnosis of disease, and treatment may be initiated without detection or quantification of expression of SNV markers associated with lymphatic abnormalities.

[0113] In other embodiments, treatment for lymphatic abnormalities may be based on a clinical diagnosis of the disease, and treatment may be initiated if expression of SNV markers associated with lymphatic abnormalities is unchanged from a control.

[0114] In some embodiments, the patient is treated without a SNV in PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, or CBL.

[0115] In some embodiments, the inhibitors are MEK1 / 2 inhibitors, which inhibit the mitogen-activated protein kinase enzymes MEK1 and / or MEK2, which can be used to affect the MAPK / ERK pathway, which is often overactive in several cancers and other disorders.

[0116] In some embodiments, the co-administered agent(s) is rapamycin or BEZ-235 (dactolisib). Rapamycin, an mTOR inhibitor, is also known as sirolimus. BEZ-235, also known as dactolisib or NVP-BEZ235, is a compound with known activity against p110, PI3K, and mTOR.

[0117] In some embodiments, the agent administered in the treatment method is rapamycin (sirolimus), everolimus (RAD001), AZD8055, temsirolimus (CCI-779, NSC 683864), KU-0063794, MHY1485, BEZ235 (NVP-BEZ235, dactolisib), PI-103, tolquinib (PP242), tacrolimus (FK506), ridaforolimus (deforolimus, MK-8669), INK 128 (MLN0128), voxatalisib (SAR245409, XL765), Torin1, omipalisib (GSK2126458, GSK458), OSI-027, PF-04691502, apitolisib (GDC-0980, RG7422), GSK1059615, jedatolisib (PF-05212384, PKI-587), WYE-354, AZD2 014, Torin2, WYE-125132 (WYE-132), PP121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388, zotarolimus (ABT-578), tacrolimus (FK506), BGT226 (NVP-BGT226), Palomido 529 (P529), chrysophanic acid, and the like.

[0118] In some embodiments, the agent administered is a MEK inhibitor selected from selumetinib (AZD6244), PD0325901, trametinib (GSK1120212), PD184352 (CI-1040), pimasertib (AS-703026), TAK-733, AZD8330, binimetinib (MEK162, ARRY-162, ARRY-438162), SL-327, rifametinib (RDEA119, Bay86-9766), and cobimetinib (GDC-0973, RG7420).

[0119] Combinations of the above-mentioned drugs can also be used in the therapeutic methods described herein to treat lymphatic abnormalities. In some embodiments, the following combinations can act additively or synergistically to treat lymphatic abnormalities, including GLA and LAM. In certain embodiments, the combinations for administration are selected from: 1) ridaforolimus and trametinib; 2) ridaforolimus and selumetinib or cobimetinib; 3) BEZ235 and selumetinib; 4) omipalisib and selumetinib or trametinib; 5) everolimus and trametinib or selumetinib; 6) sirolimus, ridaforolimus and selumetinib; 7) sirolimus, ridaforolimus and trametinib; 8) torquinib and trametinib; 9) BEZ235, torquinib and trametinib; 10) sirolimus, gedatricib and trametinib.

[0120] In some embodiments, treatment with the agents described herein is used in combination with one or more of systemic chemotherapy, interferon alpha, radiation therapy, and / or surgery.

[0121] Methods for identifying additional useful therapeutic agents Because the SNVs identified here are associated with the pathogenesis of lymphatic abnormalities, methods to identify agents that modulate the activity of genes containing such SNVs and their encoded products should lead to the generation of effective therapeutic agents for the treatment of this condition.

[0122] The chromosomal regions described herein include regions that code for proteins, and these regions can be used to rationally design therapeutics that modulate the activity of the proteins. Small peptide molecules corresponding to these regions can be advantageously used in designing therapeutics that effectively modulate the activity of the encoded proteins.

[0123] Molecular modeling should allow identification of specific organic molecules capable of binding to the active site of the protein encoded by the nucleic acid containing the SNV, based on key amino acid residues required for conformation and function. Combinatorial chemistry techniques are used to identify molecules with the greatest activity, and these molecules are iteratively developed for further cycles of screening. In one embodiment, candidate drugs can be screened from large libraries of synthetic or natural compounds. An example is an FDA-approved library of compounds available for human use.Additionally, compound libraries are available from Maybridge Chemical Co. (Trevally, Cornwall, UK), Comgenex (Princeton, NJ), Microsource (New Milford, CT), Aldrich (Milwaukee, WY), AKos Consulting and Solutions GmbH (Basel, Switzerland), Ambinter (Paris, France), Asinex (Moscow, Russia), Aurora (Graz, Austria), BioFocus DPI, Switzerland, Bionet (Camelford, UK), ChemBridge, (San Diego, CA), ChemDiv, (San Diego, CA), Chemical Block Lt, (Moscow, Russia), ChemStar (Moscow, Russia), and Exclusive Chemistry, Ltd. (Obninsk, Russia), Enamine (Kiev, Ukraine), Evotec (Hamburg, Germany), Indofine (Hillsborough, New Jersey), Interbioscreen (Moscow, Russia), Interchim (Monturcon, France), Life Chemicals, Inc. (Orange, Connecticut), Microchemistry Ltd. (Moscow, Russia), Otava, (Toronto, Ontario), PharmEx Ltd. (Moscow, Russia), Princeton Biomolecular (Monmouth Junction, New Jersey), Scientific Exchange (Center Ossipee, New Hampshire), Specs (Delft, Netherlands), TimTec (Newark, Delaware), Toronto Research Corp. (North York, Ontario), UkrOrgSynthesis (Kiev, Ukraine), Vitas-M, (Moscow, Russia), Zelinsky Institute, (Moscow, Russia), and It is commercially available from a number of companies, including but not limited to Bicoll (Shanghai, China).

[0124] Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are commercially available or can be readily prepared by methods well known in the art. It is proposed to assay compounds isolated from natural sources such as animals, bacteria, fungi, plants such as leaves and bark, and marine samples as candidates for the presence of potentially useful pharmaceuticals. It will also be understood that the pharmaceuticals screened may be derived or synthesized from chemical compositions or man-made compounds. Several commercially available libraries can be used for screening.

[0125] The polypeptides or fragments used for drug screening may be free in solution, attached to a solid support, or present intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells stably transformed with a recombinant polynucleotide expressing the polypeptide or fragment, preferably in a competitive binding assay. Such cells, either in viable or fixed form, can be used for standard binding assays. For example, the formation of a complex between the polypeptide or fragment and the agent being tested can be determined, or the extent to which the formation of a complex between the polypeptide or fragment and a known substrate is disrupted by the agent being tested can be examined.

[0126] Further techniques for drug screening include using host eukaryotic cell lines, cells (such as endothelial cells) or whole animal models (e.g., transgenic mice or zebrafish) with non-functional or altered lymphatic abnormality-related genes. In some cases, the transgenic organisms have c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R mutations in the PTPN11 gene, a c.35G>A:pG12D in KRAS, a c.1403T>C:pF468S in the BRAF gene, ac.2536G>A:pE846K in the SOS1 gene, compound mutations including c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene, or one or more of the mutations shown in Table 3. These host cell lines, cells or transgenic animals are defective at the polypeptide level. The host cell lines or cells are cultured in the presence of a drug compound. For example, in a zebrafish model, rescue of tail and / or D / V vasculature can be assessed. Additionally, induction of mTOR phosphorylation in the host cell lines can be assessed.

[0127] An exemplary method of drug screening is a method of identifying agents that alter cell signaling, such as those listed in Tables 1-2. The method would include providing a cell expressing at least one nucleic acid that includes at least one lymphatic abnormality-associated SNV, providing a cell expressing a cognate wild-type sequence corresponding to the lymphatic abnormality-associated SNV, contacting the cell expressing at least one lymphatic abnormality-associated SNV and the cell expressing the cognate wild-type sequence with a test agent, and analyzing whether the test agent alters cell signaling.

[0128] Host cells expressing lymphatic abnormality-associated SNVs or functional fragments thereof of the present invention provide a system for screening potential compounds or drugs for their ability to modulate the development of lymphatic abnormalities. Thus, in one embodiment, the nucleic acid molecules of the present invention can be used to generate recombinant cell lines for use in assays to identify agents that modulate aspects of abnormal MAPK signaling associated with lymphatic abnormalities and abnormal angiogenesis. Also provided herein are methods for screening compounds that can modulate the function of proteins encoded by SNV-containing nucleic acids.

[0129] Another method is to use a phage display library designed to express on the phage surface fragments of polypeptides encoded by nucleic acids containing SNVs. Such libraries are then contacted with combinatorial chemical libraries under conditions in which binding affinity between the expressed peptides and components of the chemical library is detected. U.S. Patent Nos. 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays.

[0130] In another embodiment, the availability of lymphatic abnormality-associated modified nucleic acids allows the production of strains of laboratory mice carrying modified nucleic acids of the invention. These lymphatic abnormality-associated modified nucleic acids correspond to compound mutations including c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R in the PTPN11 gene, a c.35G>A:pG12D in KRAS, a c.1403T>C:pF468S in the BRAF gene, ac.2536G>A:pE846K in the SOS1 gene, c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene, or any of the other mutations shown in Table 3. Transgenic mice expressing lymphatic abnormality-associated mutations of the invention provide a model system to study the role of the proteins encoded by the mutated nucleic acids in the development and progression of lymphatic abnormalities. Methods for introducing transgenes into laboratory mice are known to those skilled in the art. There are three common methods: 1. Incorporation of a retroviral vector encoding a foreign gene of interest into early embryos; 2. Injection of DNA into the pronuclei of newly fertilized eggs; and 3. Incorporation of genetically engineered embryonic stem cells into early embryos. The creation of the above transgenic mice allows the molecular elucidation of the role of target proteins in various processes related to lymphatic abnormality phenotypes. Such mice provide an in vivo screening tool for studying putative therapeutic agents in a whole animal model and are encompassed by the present invention.

[0131] As used herein, the term "animal" includes all vertebrate animals, except humans. It also includes individual animals at all stages of development, including embryonic and fetal stages. "Transgenic animal" refers to an animal that contains one or more cells that have genetic information that has been altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination, microinjection, or infection with a recombinant virus. The term "transgenic animal" does not encompass classical crossbreeding or in vitro fertilization, but rather encompasses animals in which one or more cells have been altered by or received a recombinant DNA molecule. The molecule may be targeted to a specific locus, randomly integrated into a chromosome, or may be extrachromosomally replicated DNA. "Germline transgenic animal" refers to a transgenic animal in which a genetic modification or genetic information has been introduced into a germline cell, thereby conferring the ability to transmit the genetic information to offspring. If the offspring in fact possess some or all of the alteration or genetic information, the offspring are also transgenic animals.

[0132] The altered genetic information may be foreign to the recipient's species, may be foreign only to the particular recipient individual, or may be genetic information already possessed by the recipient. In the latter case, the altered or introduced gene may be expressed differently than the native gene. Such altered or foreign genetic information may encompass the introduction of an altered lymphatic abnormality-associated nucleotide sequence.

[0133] The DNA used to modify the target gene can be obtained by a wide variety of techniques, including, but not limited to, isolation from genomic sources, preparation of cDNA from an isolated mRNA template, direct synthesis, or a combination thereof.

[0134] One type of cell that can be introduced is the embryonic stem cell (ES). ES cells can be obtained from preimplantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069). Transgenes can be efficiently introduced into ES cells by standard techniques such as DNA transfection or retrovirus-mediated introduction. The resulting transformed ES cells can then be combined with a blastocyst of a non-human animal. The introduced ES cells then colonize the embryo and contribute to the germ line of the resulting chimeric animal.

[0135] One approach to the problem of determining the contribution of individual genes and their expression products is to use isolated mutant lymphoid abnormality-associated genes as insertion cassettes to selectively inactivate wild-type genes in totipotent ES cells (as described above) and then generate transgenic mice. The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice has been described and reviewed elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al., (1992) Bio / Technology 10:534-539).

[0136] There is a technique to inactivate or change any gene region to a desired mutation by using targeted homologous recombination, which inserts a specific change into a chromosomal allele. However, compared with extrachromosomal homologous recombination, which occurs at a frequency close to 100%, homologous recombination between a plasmid and a chromosome occurs at a frequency of 10 -6 ~10 -3 It has been reported that nonhomologous plasmid-chromosome interactions are detected at a frequency of 10 times higher than the equivalent homologous insertion. 5 Times to 10 2 occurs twice as often.

[0137] To overcome this low rate of targeted recombination in mouse ES cells, various strategies have been developed to detect or select rare homologous recombinants. One approach to detect homologous alteration events is to use polymerase chain reaction (PCR) to screen a pool of transformed cells to detect homologous insertions, followed by screening individual clones. Positive genetic selection methods are also developed that construct marker genes that are activated only when homologous insertions occur, allowing these recombinants to be directly selected. One of the most powerful approaches developed to select homologous recombinants is the positive-negative selection (PNS) method, which was developed for genes where no direct selection for modifications exists. The PNS method is efficient when targeting genes that are not expressed at high levels because the marker genes have their own promoters. Non-homologous recombinants are selected against non-homologous insertions using the herpes simplex virus thymidine kinase (HSV-TK) gene with effective herpes drugs such as ganciclovir (GANC) or (1-(2-deoxy-2-fluoro-BDarabinofuranosyl)-5-ioduo-uracil, (FIAU). This counter-selection allows for an increase in the number of homologous recombinants among the surviving transformants. Utilizing a mutated lymphoid abnormality-associated nucleic acid as a target insertion cassette provides a means of detecting successful insertion, as visualized, for example, by acquisition of immunoreactivity to antibodies immunologically specific for a polypeptide encoded by the EPHB4 nucleic acid, thus facilitating screening / selection of ES cells with the desired genotype.

[0138] As used herein, a knock-in animal is, for example, an animal in which an endogenous mouse gene is replaced with a human lymphatic abnormality-associated gene of the present invention, and such a knock-in animal provides an ideal model system for studying the development of lymphatic abnormalities.

[0139] As used herein, expression of a mutated lymphoid abnormality-associated nucleic acid, a fragment thereof, or a lymphoid abnormality-associated fusion protein can be targeted in a "tissue-specific" or "cell type-specific" manner using a vector in which a nucleic acid sequence encoding all or a portion of a lymphoid abnormality-associated nucleic acid is operably linked to a regulatory sequence (e.g., a promoter and / or enhancer) that directs expression of the encoded protein in a particular tissue or cell type. Such regulatory elements can be advantageously used for both in vitro and in vivo applications. Promoters for directing tissue-specific proteins are well known in the art and described herein. Alternatively, the transgene can be under the control of an inducible promoter that functions in a tissue-specific or "systemic" manner.

[0140] The nucleic acid sequences encoding lymphoid abnormality associated variants of the invention may be operably linked to a variety of different promoter sequences for expression in transgenic animals, including, but not limited to, prion gene promoters such as the Thy-1 promoter in hamsters or mice, the PGK promoter, or the CMV promoter to express the transgene in the desired cell type.

[0141] Methods of using the transgenic mice of the invention are also provided herein. Transgenic mice into which a nucleic acid comprising a mutated lymphatic abnormality-associated nucleic acid or its encoded protein has been introduced are useful, for example, for developing methods of screening therapeutic agents to identify those capable of modulating the development of lymphatic abnormalities.

[0142] Detection Products & Kits The present invention includes compositions or products useful for detecting lymphatic abnormality SNV. For example, the nucleic acid containing lymphatic abnormality SNV, the vector expressing the nucleic acid, the marker protein containing lymphatic abnormality SNV, and the anti-lymphatic abnormality specific marker antibody are products that can detect SNVs such as c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R of the PTPN11 gene, a c.35G>A:pG12D of the KRAS gene, a c.1403T>C:pF468S of the BRAF gene, ac.2536G>A:pE846K of the SOS1 gene, and c.1236+4A>G and c.289T>G:p.C97G of the ITGA9 gene. Nucleic acid probes of sufficient length and characteristics to detect SNVs such as c.1504T>G:pS502A, c.1510A>G:pM504V, and / or c.1507G>C:pG503R of the PTPN11 gene, a c.35G>A:pG12D of KRAS, a c.1403T>C:pF468S of the BRAF gene, ac.2536G>A:pE846K of the SOS1 gene, and compound mutations including c.1236+4A>G and c.289T>G:p.C97G of the ITGA9 gene are included. The detection product may be detectably labeled.

[0143] Any product useful for detecting lymphatic abnormality-associated SNVs can be incorporated into the kit. Any product useful for treating lymphatic abnormality can be incorporated into the kit. Kits that include such detection and treatment products are included. The kit can include one or more of lymphatic abnormality-associated SNV-specific marker polynucleotides or one or a collection of such markers immobilized on a solid support, a gene chip, an oligonucleotide, a polypeptide, a peptide, an antibody, a label, a marker, a reporter, a pharma- tically acceptable carrier, a physiologically acceptable carrier, instructions, a container, a container for administration, an assay substrate, or any combination thereof.

[0144] The following examples are provided to illustrate certain embodiments of the invention, and are not intended to limit the invention in any way. EXAMPLES

[0145] Example I CCLA, like other lymphatic malformations, develops as a result of congenital lymphatic hypoplasia. To identify the genetic basis of CCLA, we performed whole-exome sequencing on DNA samples from seven patients. All of these patients had dysmorphic lymphatic vessels imaged by dynamic contrast magnetic resonance lymphography (DCMRL), a new advanced imaging technique, allowing for a better diagnosis of this group. Five patients were examined for missense, nonsense, splice change, and coding indel mutations that could potentially explain the phenotype. Findings were filtered to exclude synonymous mutations, mutations with a minor allele frequency (MAF) >0.5%, and mutations already identified in control groups in our in-house exome mutation database. Relevant candidates were subjected to manual curation. As a result, we identified one somatic mutation and five de novo missense mutations in four genes (PTPN11, KRAS, BRAF, SOS1) involved in the Ras / MAPK signaling pathway (Table 3). Furthermore, two heterozygous mutations, c.1236+4A>G and c.289T>G:p.C97G, were found in ITGA9 in one patient with primary lymphedema and retrograde lymphatic flow. ITGA9 encodes intergrin α9, a cell surface glycoprotein that mediates cell-cell adhesion and cell-matrix interactions. Integrin α9 binds VEGF-C, and inactivation of ITGA9 leads to thoracic cage formation and inhibits lymphatic valve formation in mice. The sequences of these proteins are listed in the NCBI database and are as follows: a) c.1504T>G:pS502A in PTPN11; NCBI reference sequence present in S502::NP_002825.3 b) c.1510A>G:pM504V in PTPN11 NCBI reference sequence present in M504: NP_002825.3 c) c.1507G>C:pG503R in PTPN1 NCBI reference sequence present in G503: NP_002825.3 d) KRAS c.35G>A:pG12D NCBI reference sequence present in G12: NP_203524.1 e) BRAF c.1403T>C:pF468S NCBI reference sequence present in F468: NP_004324.2 f) c.2536G>A:pE846K in SOS1 NCBI reference sequence present in E846: NP_005624.2 g) ITGA9 c.1236+4A*>G and c.289T>G: p.C97G NCBI reference sequence present in pC97: NP_002198.2

[0146] A variant located 4 bp away from an exon-intron junction and also present within an intron [Table 3] TIFF2025020109000009.tif49141TIFF2025020109000010.tif37141TIFF2025020109000011.tif206141TIFF2025020109000012.tif196141TIFF2025020109000013.tif206141TIFF2025020109000014.tif199141TIFF2025020109000015.tif194141TIFF2025020109000016.tif197141TIFF2025020109000017.tif210140TIFF2025020109000018.tif197141TIFF2025020109000019.tif210139TIFF2025020109000020.tif199141TIFF2025020109000021.tif210141TIFF2025020109000022.tif210140TIFF2025020109000023.tif197141TIFF2025020109000024.tif196141TIFF2025020109000025.tif210139TIFF2025020109000026.tif205141TIFF2025020109000027.tif64141

[0147] RASopathies are a group of genetically heterogeneous disorders including Noonan syndrome (NS), Cardiofaciociocutaneous syndrome (CFC), Neurofibromatosis type 1 (NF1), Noonan syndrome with multiple lentigines (NSML), Costello syndrome (CS), Legius syndrome, and Legius syndrome, which have mutations in the Ras / MAPK pathway involving PTPN11, SOS1, RAF1, KRAS, HRAS, MAP2K1, MAP2K2, NRAS, CBL, SHOC2, BRAF, RIT1, A2ML1, SPRED1, and NF1. Lymphatic defects in NS have been reported in a subset of NS patients, but vary in severity, location, and time of onset. After reviewing the literature, we identified a total of 52 prenatal and postnatal patients who presented with clinical features of Noonan or Noonan-like syndrome and lymphatic deficiency, including pleural effusion, pericardial effusion, pleural effusion, edema, lymphangiectasia, and lymphedema. We identified mutations in PTPN11 that explain their disease. Thus, therapeutic intervention in the ras / MAPK ERK pathway should be able to reverse the disease phenotype in these patients, similar to what we have shown so far for ARAF gene mutations. Thus, our finding that patients with central lymphatic malformations have germline or somatic mutations in the Ras / MAPK pathway is novel and identifies a new target for therapeutic development. Cellular and model data supporting these RAS / MAPK pathways are presented below.

[0148] We developed a cellular model to investigate the effects of mutations that may activate ERK by retrovirally expressing wild-type and mutant proteins in the cell line Ea.hy926. The Ea.hy926 cell line is a fusion between human umbilical vein endothelial cells and a melanoma cell line. The Ea.hy926 cell line is an immortalized fusion between human umbilical vein endothelial cells and a melanoma cell line, and retains some characteristics of endothelial cells. Using this cell line, we successfully demonstrated the cellular effects of mutations in the ARAF gene, which are responsible for central lymphatic abnormalities. We also successfully reversed the effects of mutant ARAF in the same cell line using the MEK inhibitor trametinib.

[0149] Figure 1 shows the levels of activated ERK in EA.hy926 cells expressing WT or mutant BRAF or PTPN11 proteins. When normalized for protein abundance, cells expressing mutant BRAF in this experiment contain more than five-fold more phosphorylated ERK than cells expressing wild-type BRAF. Mutant BRAF is more highly expressed than WT, as previously observed with ARAF.

[0150] In Figure 2, changes in cell morphology can be observed in cells expressing the F486S BRAF mutant. Filamentous actin, as revealed by phalloidin staining, is present throughout the cell body in cells expressing wild-type BRAF. However, in cells expressing the BRAF mutant, actin is largely restricted to the periphery. This behavior is consistent with previous observations of cells expressing ARAF-mutants. VE-cadherin staining is largely intracellular in cells expressing either WT or mutant forms of BRAF. Previous results with ARAF suggest that expression of WT BRAF also induces ERK activation in the Ea.hy926 cell line. This results in internalization of VE-cadherin, although WT BRAF does not alter the overall cell morphology. Figure 3 highlights the impact of ERK activation on both VE-cadherin localization and cell morphology. Treatment of cells expressing WT or mutant BRAF dramatically increases cell surface staining of VE-cadherin and increases filamentous actin in the cell body. In this experiment, the morphological differences between WT and mutant-expressing cells were less pronounced, but this may represent variability between experiments or may be due to the increased culture time and cell density in this experiment compared to Figure 2. No changes in cell morphology or distribution of VE-cadherin were observed in cells expressing mutant PTPN11.

[0151] To find inhibitors with better therapeutic effects, we treated cells expressing WT and mutant BRAF or PTPN11 with various MEK inhibitors (see Figure 4 and Figure 5). We performed titration curves with PD0352971, pimasertib, and rifametinib, with trametinib as a comparison. As expected, each inhibitor was able to reduce pERK levels in cells in a dose-dependent manner. Note that in our experiments, mutant PTPN11 did not appear to activate ERK above the levels seen with WT PTPN11. BRAF F486S consistently induces ERK activation above WT levels, although the specific fold increase varies from gel to gel.

[0152] Zebrafish models of lymphatic disease Clones were transduced using the Gateway system (Invitrogen, Kwan 2007, Villefranc 2007 17937395, 17948311) into a vector with flanking sites for the Tol2 transposase to allow integration into the genome (Kawakami 1999, 10564832). Approximately 10 pl of 25 ng / ul Tol2 mRNA and vector DNA were injected into the first cell of fresh fertilized eggs. Casperfish expressing mrc1a:GFP were injected with the constructs to visualize lymphatic vessels. Confocal scanning was performed using a Zeiss LSM710 confocal microscope and Zen software. Confocal Z-stack images were overlaid using the maximum intensity projection function of the Zeiss Zen software. Images were edited with imageJ (Fiji) {Schindelin 2012} and Powerpoint (Microsoft).

[0153] To assess whether mutant human KRAS G12D affects lymphatic development in zebrafish, we cloned both mutant and WT genes behind the lymphatic vessel-specific mrc1a promoter (Jung 2017, PMID:28506987) and linked to mCherry fluorophore at the v2a autocatalytic cleavage site (Provost 2007,17941043). Genomic integration was achieved using flanking Tol2 transposase sites. Injection of this construct with tol2 mRNA resulted in small patches of lymphatic vessel cells expressing the transgene and mCherry marker in most injected fish when phenotyped at 7 dpf (days post fertilization).

[0154] We examined the phenotype of the trunk thoracic duct, which is one of the largest blood vessels at this stage and in which expression of the transgenic clones was most consistent. WT expression of KRAS had no effect on lymphatic development, but KRAS G12D caused the thoracic duct to expand and dilate, and appear to merge with the ventral cardinal vein.

[0155] Additional studies have identified further genes / mutations in the RAS / MAPK pathway that explain lymphatic abnormalities and provide new therapeutic intervention opportunities. As shown in Table 3, we identified germline or somatic mutations in RASA1, RAF1, RIT1, NF1, CBL1, and BRAF, which are involved in the RAS / MAPK signaling pathway, further supporting the common genetic etiology of these disease groups and the importance of RAS / MAPK pathway mutations in lymphatic malformations. In addition, one homozygous missense mutation was found in a patient with lymphedema and impaired lymphatic conduction. In this study, we present data showing that therapeutic intervention in the RAS / MAPK pathway can reverse the elevated ERK1 / 2 activity and lymphatic phenotype induced by this mutation in zebrafish, similar to what we have previously shown for ARAF mutations.

[0156] We developed a cellular model to better understand and study the effects of potential ERK-activating mutations by using primary lymphatic endothelial cells instead of established cell lines, capturing the characteristics of mutations in disease-relevant cell types. We retrovirally expressed wild-type and mutant proteins in human dermal lymphatic endothelial cells (HDLECs). Using these cells, we demonstrated that mutations in the ARAF gene cause central lymphatic abnormalities. We also successfully reversed the effects of mutant ARAF using the MEK inhibitor trametinib, and our results were recently published in Nature Medicine. Furthermore, seven additional MEK / ERK inhibitors were tested in this model and showed biochemical and morphological reversal of the ARAF mutation-induced effects comparable to trametinib, including PD0325901 (Figure 6), CI1040 / PD184352 (Figure 7), pimasertib (Figure 8), TAK-733 (Figure 9), AZD8330 (Figure 10), rifametinib (Figure 11), and ulixertinib (Figure 12).

[0157] Figures 6-11 show the effect of different drugs (as indicated in the figures) on HDLECs expressing the ARAF mutant S214P. All drugs tested were sufficient to reverse the morphological differences induced by ARAF-S214P, showing greater accumulation of VE-cadherin at the cell membrane. Green is HA staining for ARAF-expressing cells, and red is VE-cadherin staining.

[0158] Ulixertinib is an orally active ERK1 / 2 inhibitor. Previous studies have revealed that p-ERK1 / 2 levels are increased in various cancer cell lines after ulixertinib treatment. However, phosphorylation of RSK (a substrate of ERK1 / 2 protein) is decreased, which is consistent with sustained ERK1 / 2 inhibition. As shown in the top panel of Figure 12, ulixertinib treatment suppresses phosphorylation of RSK3, an ERK substrate. Also, as shown in the bottom panel, ulixertinib can rescue the loss of VE-cadherin from the cell-cell junctions of HDLECs expressing ARAF-S214P by almost completely restoring it at a concentration of 300 nM (Figure 12; green indicates HA staining for ARAF-expressing cells, red indicates VE-cadherin staining, and white indicates DAPI staining for nuclei). Similarly, expression of BRAF-F486S in HDLECs results in a notable lack of VE-cadherin accumulation between adjacent cells (Figures 13-14), essentially resulting in a phenotype similar to that of ARAF mutations. As expected, ulixertinib (Figure 13) or trametinib (Figure 14) treatment can rescue the phenotype, with trametinib (Figure 14 top panel) suppressing the elevation of ERK phosphorylation. Furthermore, HDLECs expressing RAF1 or KRAS mutations showed similar morphological changes and similarly elevated p-ERK, which could be normalized by trametinib and ulixertinib (Figures 15-16). On the other hand, RIT1 mutations induce weak activation of p-ERK (Figure 17).

[0159] We utilized spheroid sprouting assays (3D lymphangiogenesis assays) with HDLECs to gain insight into the impact of the mutations on lymphangiogenesis. HDLECs expressing EPHB4-ins, EPHB4-R763Q, or EPHB4-K885 show enhanced lymphangiogenic capacity compared to HDLECs expressing EPHB4-WT, as measured by sprout length in 3D lymphaspheroid sprouting assays performed in the presence of vascular endothelial growth factor C (VEGFC) with or without ephrin B2 (Figure 18, upper and lower left panels). Both rapamycin and OSI-027, potent and selective inhibitors of mTORC1 and mTORC2, are able to rescue the increased sprouting of the mutants (Figure 18, lower right panel).

[0160] In a zebrafish model, overexpression of KRAS-G12D mutant, but not wild-type KRAS, in lymphatic vessels led to dilated thoracic ducts and significant edema. We further investigated whether either mTOR or MEK / ERK inhibitors could rescue the phenotype. Unlike rapamycin, CI1040, and SL-327, which showed no significant effect, treatment with MEK inhibitors greatly improved the phenotype. Specifically, treatment with cobimetinib, pimasertib, TAK-733, AZD8330, and PD0325901 reduced edema and improved disorganized lymphatic branching (Figure 19). Furthermore, treatment with NVP-BEZ235, a dual inhibitor of PI3K and mTOR, significantly reduced edematous larvae (Figure 19).

[0161] We also overexpressed PTPN11 mutations in a zebrafish model. Figure 20 shows that mosaic expression of the S502A or G503R mutations of PTPN11 resulted in mild lymphatic abnormality phenotypes, such as lymphatic expansion and misdirection, and fusion of the posterior cardiac vein and thoracic duct.

[0162] Zebrafish have two RASA1 homologs, rasa1a and rasa1b. We designed gRNAs targeting both the rasa1a and rasa1b genes and injected them into Cas9 transgenic embryos, resulting in the formation of large edema (Figure 21).

[0163] References for Example I 1.Alitalo K, Tammela T, Petrova TV 2005 Lymphangiogenesis in development and human disease. Nature 438:946-953 2.Trenor CC,3rd, Chaudry G 2014 Complex lymphatic anomalies.Semin Pediatr Surg 23:186-190 3.Levine C 1989 Primary disorders of the lymphatic vessels--a unified concept.J Pediatr Surg 24:233-240 4.Wassef M,Blei F,Adams D,Alomari A,Baselga E, Berenstein A, Burrows P,Frieden IJ,Garzon MC,Lopez-Gutierrez JC,Lord DJ,Mitchel S,Powell J,Prendiville J,Vikkula M 2015 Vascular Anomalies Classification:Recommendations From the International Society for the Study of Vascular Anomalies.Pediatrics 136:e203-214 5.Hilliard RI, McKendry JB, Phillips MJ 1990 Congenital abnormalities of the lymphatic system: a new clinical classification. Pediatrics 86:988-994 6.Smeltzer DM,Stickler GB,Fleming RE 1986 Primary lymphatic dysplasia in children:chylothorax,chylous ascites, and generalized lymphatic dysplasia.Eur J Pediatr 145:286-292 7.Faul JL,Berry GJ, Colby TV,Ruoss SJ,Walter MB,Rosen GD,Raffin TA 2000 Thoracic lymphangiomas,lymphangiectasis,lymphangiomatosis,and lymphatic dysplasia syndrome.Am J Respir Crit Care Med 161:1037-1046 8.Brouillard P, Boon L, Vikkula M 2014 Genetics of lymphatic anomalies. J Clin Invest 124:898–904 9.Luks VL,Kamitaki N,Vivero MP, Uller W, Rab R,Bovee JV,Rialon KL,Guevara CJ,Alomari AI,Greene AK,Fishman SJ,Kozakewich HP,Maclellan RA,Mulliken JB,Rahbar R,Spencer SA,Trenor CC,3rd,Upton J,Zurakowski D,Perkins JA, Kirsh A, Bennett JT, Dobyns WB, Kurek KC, Warman ML, McCarroll SA, Murillo R 2015 Somatic mutations in PIK3CA.J Pediatr 166:1048–1054 e1041–1045 10.Kurek KC, Luks VL,Ayturk UM, Alomari AI, Fishman SJ, Spencer SA, Mulliken JB, Bowen ME, Yamamoto GL, Kozakewich HP, Warman ML 2012 Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome. Am J Hum Genet 90:1108-1115 11.Lindhurst MJ, Sapp JC,Teer JK,Johnston JJ, Finn EM, Peters K,Turner J,Cannons JL, Bick D, Blakemore L, Blumhorst C,Brockmann K, Calder P, Cherman N, Deardorff MA,Everman DB,Golas G, Greenstein RM, Kato BM,Keppler-Noreuil KM, Kuznetsov SA,Miyamoto RT,Newman K, Ng D, O’’Brien K,Rothenberg S,Schwartzentruber DJ, Singhal V, Tirabosco R,Upton J,Wientroub S,Zackai EH,Hoag K,Whitewood-Neal T,Robey PG, Schwartzberg PL,Darling TN,Tosi LL,Mullikin JC,Biesecker LG 2011 A mosaic activating mutation in AKT1 associated with the Proteus syndrome.N Engl J Med 365:611-619 12.Revencu N,Boon LM,Mendola A,Cordisco MR,Dubois J,Clapuyt P,Hammer F,Amor DJ,Irvine AD,Baselga E,Dompmartin A,Syed S,Martin-Santiago A,Ades L,Collins F,Smith J,Sandadura S,Brorrow, PE,Brorrow,Cozz, VR,Cozz M,Brunetti-Pierri N,Vicente A,Abramowicz M,Desir J,Vilain C,Chung WK,Wilson A,Gardiner CA,Dwight Y,Lord DJ,Fishman L,Cytrynbaum C,Chamlin S,Ghali F,Gilaberte Y,Joss S,Boente Mdel C,Leabreeze,Drue,M.D. S,Martorell L,Gonzalez-Ensenat MA, Mazereeuw-Hautier J,O'Donnell B,Bessis D, Pyeritz RE,Salhi A,Tan OT,Wargon O,Mulliken JB,Vikkula M 2013 RASA1 mutations and associated phenotypes in 68 families with capillary malformation-malformation. Hum Mutat 34:1632–1641 13.Burrows PE,Gonzalez-Garay ML,Rasmussen JC, Aldrich MB, Guilliod R, Maus EA, Fife CE,Kwon S, Lapinski PE,King PD,Sevick-Muraca EM 2013 Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man. Proc Natl Acad Sci USA 110:8621–8626 14.Lo IF, Brewer C, Shannon N, Shorto J, Tang B, Black G, Soo MT, Ng DK, Lam ST, Kerr B 2008 Severe neonatal manifestations of Costello syndrome.J Med Genet 45:167–171 15.Fabretto A,Kutsche K,Harmsen MB,Demarini S,Gasparini P, Fertz MC,Zenker M 2010 Two cases of Noonan syndrome with severe respiratory and gastroenteral involvement and the SOS1 mutation F623I.Eur J Med Genet 53:322–324 Eur J Hum Genet 24:690–696 [ PubMed ] 16. Joyce S, Gordon K, Brice G, Ostergaard P, Nagaraja R, Short J, Moore S, Mortimer P, Mansour S 17.Morcaldi G, Bellini T,Rossi C,Maghnie M, Boccardo F, Bonioli E, Bellini C 2015 Lymphodysplasia and Kras Mutation:A Case Report and Literature Review. Lymphology 48:121–127 18.Makinen T, Adams RH, Bailey J, Lu Q, Ziemiecki A, Alitalo K, Klein R, Wilkinson GA 2005 PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev 19:397-410 19.Kume T 2010 Specification of arterial,venous,and lymphatic endothelial cells during embryonic development.Histol Histopathol 25:637-646 20.Hashimoto T,Tsuneki M,Foster TR, Santana JM,Bai H,Wang M,Hu H,Hanisch JJ,Dardik A 2016 Membrane-mediated regulation of vascular identity.Birth Defects Res C Embryo Today 108:65-84 21.Martin-Almedina S,Martinez-Corral I,Holdhus R,Vicente A,Fotiou E,Lin S,Petersen K,Simpson MA,Hoischen A,Gilissen C,Jeffery H,Atton G,Karapouliou C,Brice G,Gordon K,Wiseman JW,Wedin M,Rockson SG, Jeffery S, Mortimer PS,Snyder MP,Berland S, Mansour S,Makinen T, Ostergaard P 2016 EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis.J Clin Invest 126:3080-3088 22.Kettleborough RN,Busch-Nentwich EM,Harvey SA,Dooley CM,de Bruijn E,van Eeden F,Sealy I,White RJ,Herd C,Nijman IJ,Fenyes F,Mehroke S, Scahill C, Gibbons R, Wali N, Carruthers S, Hall A, Yen J, Cuppen E, Stemple DL 2013 A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature 496:494-497 23.Sun S,Chen S,Liu F,Wu H,McHugh J,Bergin IL,Gupta A,Adams D,Guan JL 2015 Constitutive Activation of mTORC1 in Endothelial Cells Leads to the Development and Progression of Lymphangiosarcoma through VEGF Autocrine Signaling. Cancer Cell 28:758-772. EXAMPLES

[0164] Example 2 Recurrent ARAF mutations cause central conducting lymphatic abnormalities treatable with MEK inhibitors Recent studies have demonstrated the utility of sirolimus in the treatment of generalized lymphatic malformation (GLA) and central lymphatic malformation (CCLA)3-5, but the lack of a clear clinical distinction between these disorders, due to their rarity and overlapping diagnostic criteria, hampers the development of innovative therapies6-9. GLA is defined as a multifocal lymphatic malformation present in multiple areas with micro / macrocyst lymphatic malformations and often accompanied by bone destruction9-11. CCLA, on the other hand, is a dysfunction of the thoracic duct (TD) or thoracic duct, causing lymphatic reflux or abnormal lymphatic drainage1,12,13. Both disorders present with thoracic pleural effusion, bile ascites, and lymphedema. The overlap between these seemingly distinct disorders suggests that a common pathway, rather than a common gene, is responsible. This implies a variety of clinical syndromes, suggesting that the distinction between the disorders may be artificial. Here, we report the use of whole-exome sequencing (WES) to identify recurrent missense mutations in ARAF underlying a severe, progressive lymphatic disease characterized by complex lymphatic abnormalities in two unrelated patients. This result serves as a prime example of how genetic classification can help classify complex medical diseases, resulting in biologically-based treatment that was life-saving in our case.

[0165] The following materials and methods are provided to facilitate the practice of Example II.

[0166] patient After approval from the Institutional Review Board at the Children's Hospital of Philadelphia (CHOP) and written informed consent, blood samples from the primary care physician, Proband (P1), and his parents were obtained and sequenced. Proband presented with severe lymphatic fluid collections in the chest, pericardium, abdomen, lower extremities, and genitals, and was followed and treated at the Center for Lymphatic Imaging and Interventions at CHOP. A second unrelated adult patient (P2) was recruited through the Lymphangiomatosis & Gorham's Disease Alliance (LGDA) Patient Registry, along with available family members. P1's birth and family history were unremarkable except for a capillary malformation in the left flank, and his childhood growth and development were normal. At the age of 10 years, he developed swelling in the lower abdomen, thighs, scrotum, and penis. Two months later, he visited a local hospital complaining of shortness of breath and exercise intolerance. Chest radiograph confirmed cardiomegaly, and echocardiogram confirmed a large pericardial effusion. Pericardiocentesis was performed, and 1 liter of cerebrospinal fluid was drained. Total parenteral nutrition was administered, but the effusion continued to drain, and the patient was transferred to CHOP for further management. At CHOP, dynamic contrast-enhanced magnetic resonance lymphangiography was performed as an initial evaluation, and showed a large pericardial effusion and reflux from the dilated lumbar and retroperitoneal network to the tortuous TD, which was dilated toward the left denominator vein (Fig. 1a, c, d). Note that Fig. 1b shows an image of an asymptomatic individual for reference. In addition, retrograde lymphatic flow to the liver, mesentery, penis, and scrotum, and retrograde lymphatic flow from the distal TD to the mediastinum and pericardium were observed (Fig. 1c-e). Therefore, a stent was placed in the distal TD, and lipiodol embolization was performed to block abnormal lymphatic flow to the mediastinum and pericardium. The patient was discharged one month later with stable pericardial effusion, but soon afterwards developed respiratory distress due to massive fluid reaccumulation and required supplemental oxygen (up to 5 liters delivered via nasal cannula).Sirolimus was initiated at a dose of 2.5 mg daily based on well-tolerated trough levels, resulting in a median trough level of 11.8 (range 6.8-16.2 μg dl-1) between May and November 2016.Over a 1.5-year period, the patient underwent multiple percutaneous interventions and surgical lymphatic procedures, including repeated thoracentesis and pleural drainage, multiple percutaneous lymphatic embolizations, two bilateral surgical pleural punctures, surgical lymphatic anastomoses of the thigh, abdomen, and retroperitoneum, and surgical ligation and embolization of the inguinal lymphatics due to worsening penile and scrotal edema. Despite multiple attempts to control the pericardial effusion, lymphedema of the penis, scrotum, lower extremities, and lower abdomen worsened, and the patient's symptoms continued to worsen, leading to consideration of palliative care. Following the last procedure, sirolimus was discontinued 5 months prior to trametinib. [Table 4]

[0167] Patient P2 is an unrelated adult female who was diagnosed with lymphangiomatosis at age 31 years. P2 had been symptomatic for many years prior to diagnosis, with prominent pulmonary infiltrates and multiple pleural punctures prior to pleural adhesions. She also had extensive gastrointestinal involvement, requiring a specialized low-fat diet and medium-chain triglyceride oil supplementation, as well as intermittent total parenteral nutrition. As her symptoms continued of unknown etiology, computed tomography and magnetic resonance imaging were performed, which were consistent with lymphangiomatosis involving the kidneys, liver, spleen, and lungs.

[0168] A liver biopsy confirmed lymphangiomatosis. The patient was further treated with albuterol and diuretics and used a power scooter for fatigue and dyspnea. No bone involvement was identified. The patient was recruited from the Lymphangiomatosis & Gorham's Disease Alliance and was not local, so was lost to follow-up and died from complications related to his underlying lymphatic disease, making him unable to undergo trials of other treatments.

[0169] WES and bioinformatics analysis Exome data were examined for missense, nonsense, splice variations, and coding indels consistent with dominant or recessive genetic models. Results were filtered to exclude variants with the following components: synonymous variants, variants in known pseudogenes, variants with minor allele frequency (MAF) >0.5% in any of the 6,503 exomes from the 1000 Genomes Project or the National Heart, Lung, and Blood Institute Exome Sequencing Project (ESP6500SI), and variants already identified in controls in our in-house exome variant database. Genes were then prioritized based on fulminant prediction and biological relevance by consulting the Online Mendelian Inheritance in Man database.

[0170] Expression and characterization of ARAF mutations in mammalian cell lines HEK293T and HeLa cells were obtained from the American Type Culture Collection and cultured at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Primary adult HDLECs were obtained from Promocell and cultured in Endothelial Cell Growth Medium MV 2 (Promocell) according to the manufacturer's instructions. Full-length ARAF cDNA (plasmid no. 23725)41 obtained from Addgene was amplified from its original vector and cloned as a BamHI / XhoI fragment into the pcDNA3.1 vector containing two copies of a FLAG tag (DDKDDK) followed by two copies of a STREP tag (WSHPQFEK). The S214P mutation was created by site-directed mutagenesis using the NEB Q5 mutagenesis kit according to the manufacturer's instructions. Transfections into HEK293T and HeLa were performed using Fugene HD (Promega) with 3 μg of DNA (empty vector, WT ARAF (ARAF-WT) or ARAF mutant (ARAF-S214P)) and 9 μl of transfection reagent according to the manufacturer's protocol. Thirty-six to 48 hours after transfection, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice using freshly ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 100 mM MgCl. Cells were lysed using fresh ice-cold cell lysis buffer containing 4,100 mM NaCl, 50 mM β-glycerophosphate, 10% glycerol (w / v), 1% NP-40 (w / v), 1 mM EDTA, 2 mM NaVO4, and complete EDTA-free protease inhibitor cocktail (Roche Applied Science) at 20 μl per ml of lysis buffer. After clearing the cell lysate by centrifugation, the supernatant was collected and used for Western blotting or for immunoprecipitation using Anti-FLAG M2 Affinity Gel (cat. no. A2220, Sigma) followed by Western blotting.Immunoprecipitates and lysates were run on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific) and blotted with primary antibodies including anti-phospho-p70S6K-Thr389 (cat.no.9205S, Cell Signaling Technology; 1:1,000), anti-phospho-mTOR Ser2448 (cat.no.5536P, Cell Signaling Technology; 1:1,000), anti-FLAG (cat.no.F3165, Sigma; 1:4,000), anti-phospho-p38 Thr180 / Tyr182 (cat.4511, Cell Signaling Technology; 1:1,000), anti-PAN-14-3-3 (cat.no.sc-629, Santa Cruz Biotechnology; 1:500), and anti-phospho-Akt-Ser473 (cat.no.4060, Cell Signaling Technology; 1:500). Antibodies used were anti-phosphop44 / 42-(Erk1 / 2)-Thr202 / Tyr204 (cat. no. 4376, Cell Signaling Technology; 1:1,000), or anti-β-actin (cat. no. sc-69879, Santa Cruz Biotechnology; 1:1,000).

[0171] The ARAF sequence from the pCDNA3.1-F2S2-ARAF-WT or -S214P constructs shown above was cut with BamHI / XhoI and introduced into the BglII / XhoI sites of a modified pMSCV plasmid containing amino-terminal FLAG and HA tags. Virus production was performed using Fugene, with HEK293T receiving 8 μg of total DNA (pMSCV-ARAF-WT or -S214P together with envelope and packaging plasmids) and 18 μl of transfection reagent. After 72 h, the viral supernatant was harvested and filtered. The cell culture medium was replaced with viral supernatant and 8 μg ml -1HDLECs were infected by supplementing 1000 μl of polybrene and filtering through a 0.45 μm filter. Cells were spinfected at 650 g for 90 min followed by incubation for 6 h, at which point the viral supernatant was replaced with standard culture medium. Transduced HDLECs were cultured for 48 h and confirmed before use in experiments. Transduction efficiency, as observed by HA staining, ranged from 40% to 60%.

[0172] Immunofluorescence staining and western blotting of HDLECs Round (12 mm) coverslips (VWR) were coated in 24-well plates (Corning) with 0.1% gelatin in water for 10 min and then air-dried for 15 min. Transduced HDLECs were plated at 100,000 cells per well in 0.5 ml of culture medium and cultured for 48 h in the presence or absence of trametinib. Cells were washed with warm serum-free Dulbecco's modified Eagle's medium and fixed with 4% paraformaldehyde. Fixed cells were washed twice with PBS and twice with 0.1% BSA in PBS. Cells were permeabilized and blocked by incubation with 10% normal donkey serum (Jackson Immunoresearch) and 0.3% Triton X-100 (Sigma Aldrich) in PBS. VE-cadherin antibody (Thermo Fisher Scientific) was diluted in 0.01% normal donkey serum, 0.1% BSA, and 0.3% Triton X-100 (PBS) (final concentration: 2 μg ml -1)and stained for 1 h. Coverslips were washed twice with 0.1% BSA in PBS. Goat-anti-rabbit Alexa546 (Thermo Fisher Scientific; final concentration: 8 μg ml-1) and phalloidin Alexa350 (Thermo Fisher Scientific; final concentration: 5 units ml-1) were diluted in 0.01% normal donkey serum, 0.1% BSA, and 0.3% Triton X-100 in PBS and stained for 1 h. When used, HA-Tag (6E2) mouse antibody (cat. no. 2367, Cell Signaling Technology) was diluted 1:100 in 0.1% BSA and 0.3% Triton X-100 in PBS and stained for 1 h. Coverslips were washed twice with 0.1% BSA in PBS and twice with PBS. Coverslips were soaked in water to remove residual salt and mounted on slides using Prolong Gold antifade reagent (Thermo Fisher Scientific). Images were acquired using a Leica DM6000 motorized upright microscope equipped with a Photometrics HQ2 high-resolution monochrome CCD (charge-coupled device) camera and LAS AF software (Leica Microsystems). Z-stacks were acquired at 10x magnification. Images were further processed with the Fiji software package. Brightness and contrast adjustments were performed. Identical brightness and contrast settings were applied to all images. Fluorescence values ​​were measured in regions of interest (ROIs) drawn to include entire individual cells or ROIs drawn to include entire cell bodies but not cell-cell junctions. From these measurements, plasma membrane values ​​(whole cell minus intracellular) were calculated, and ratios of plasma membrane to intracellular values ​​were calculated and plotted. Cell length and width were also measured using the line tool and ROI manager. In both analyses, cells that were determined to clearly express ARAF by HA staining were analyzed 5 per 1 × 10 field. For each experiment, 5 × 10 fields were acquired per condition. Experiments were performed with cells from three independent thaws and transfections of HDLEC, for a total of 75 cells per condition.For western blotting of HDLECs with trametinib, 20,000 transduced HDLECs cells were plated in 96-well plates in the presence of increasing amounts of trametinib. Cells were cultured in the presence of drug for 24 h and then lysed with 40 mM HEPES pH 7.5, 120 mM NaCl, 0.3% CHAPS, 50 mM NaF, 1.5 mM NaVO3 and a protease inhibitor cocktail. Lysates were cleared by centrifugation at 20,000g for 5 min at 4°C. Proteins were separated on 4-12% NuPAGE Bis-Tris gels. Blotting was performed with the antibodies described above.

[0173] 3D lymphocyte spheroid sprouting assay Multicellular spheroids for lymphatic sprouting assays were generated by seeding 7,500 HDLECs expressing ARAF-WT or ARAF-S214P into wells of a 96-well plate precoated with 1.5% agarose. Under these conditions, all HDLECs aggregate into a single spheroid by 24 h. After formation, each spheroid was transferred to a gelling solution consisting of type I collagen (cat No. 354236 Corning; final concentration 354236, Corning; final concentration = 1.5 mg ml-1; pH neutralized with NaOH) and trametinib at the indicated concentrations and polymerized at 37 °C. Once solidified, Endothelial Cell Growth Medium MV2 (without VEGFC) containing the appropriate concentration of trametinib was added on top of the collagen gel. After 2 days of culture, Z-stack images of the embedded spheroids were taken with an EVOS FL Auto Imaging System (Thermo Fisher Scientific) at a step size of 8.5 μm. The number and length of capillary-like sprouts growing from each spheroid were measured using ImageJ (https: / / imagej.nih.gov / ij / ) software.

[0174] MTT proliferation assay using transformed HDLECs The proliferation of transduced HDLECs was measured using the Cell Proliferation Kit I (MTT) from Roche Applied Science. Briefly, 2 days after retroviral transduction, ARAF-WT- and -S214P-expressing HDLECs were harvested, counted, and replicated in flat-bottom 96-well plates at 10,000 cells per well in 100 μl of medium. At the indicated times after plating, 10 μl of MTT was added to the appropriate wells and incubated at 37°C for 4 hours. 100 μl of solubilization reagent was added, followed by overnight incubation at 37°C. Absorbance was measured at 550 nm and 700 nm using a Spectramax i3 Multi Mode plate reader (Molecular Devices), and A550nm-A700nm was calculated. The 4-hour time point after plating was included as a guide for when low-proliferating cells were added to the experiment.

[0175] Transgenic expression of human ARAF in zebrafish All procedures using zebrafish were approved by the Institutional Animal Care and Use Committee at CHOP (IAC 001154) and followed the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. Human mutant and WT ARAF cDNAs were cloned into the pDONR221 vector without a stop codon; a zebrafish-adapted Kozak sequence (GCAAACATGG) was used. Expression constructs were assembled using Tol2 backbone vectors containing a Gateway cloning cassette (44,45). Constructs were co-injected with Tol2 messenger RNA (46).

[0176] ARAF was expressed in veins and lymphatic vessels using the zebrafish mrc1a promoter, and expression was visualized by mCherry linked to ARAF by the cleavage site of the autocatalytic V2a protein. For imaging, larvae were mounted in low-melting agarose and multiple Z-stack images were taken with a Zeiss LSM710 confocal microscope using a 20x lens. Confocal Z-stack images were overlaid using the maximum intensity projection function of the Zeiss Zen software.

[0177] To analyze TD expansion, somites separated by intersegmental lymphatic vessels expressing the transgene in the TD were selected. Morphology was assessed as either normal (WT), moderately expanded (TDs expanded but separate from the PCV), or severely expanded (TDs and PCVs indistinguishable in Z-projection). Images were edited with ImageJ (Fiji). Each experiment was performed in triplicate, and a total of 40 animals were analyzed.

[0178] Inhibitory drug treatment in zebrafish Drug treatments were performed in 6-well plates with up to 20 larvae per group. Cobimetinib was diluted in embryo culture medium containing 0.01M Tris pH 7.2 and 0.1% DMSO. Cobimetinib was used at 1 μM.

[0179] p-ERK antibody staining in zebrafish Fish were injected as above, and larvae with prominent expression of WT or mutant ARAF / mcherry were selected for analysis. Larvae were fixed overnight in 4% paraformaldehyde solution in PBS containing Tween-20 (PBST). Larvae were washed in PBST and incubated with 2% Triton X-100 for 24 h at 4 °C. Larvae were then blocked with 10% bovine serum and stained with phospho-ERK T202 / Y204 antibody (cat.no.9101, Cell Signaling Technology, 1:200) overnight at 4 °C, washed in PBST, and stained with Alexa Fluor 488 goat anti-rabbit secondary antibody (cat.no.A11008, Thermo Fisher Scientific, 1:400).

[0180] statistics For all cell-based assays, significance was assessed by unpaired, two-tailed Student's t-test to compare two groups. Statistical analysis was performed with GraphPad Prism 7.0d software. Data were presented as dot plots with 25th–75th percentile boxes, whiskers from minimum to maximum, median-centered box and whisker plots, or bars of mean ± s.e.m. For all assays, three independent experiments were performed with independent transfections of HDLECs, except for the proliferation test, for which no statistical analysis was performed. For the 14-3-3 protein association assay, three independent experiments were performed with independent transfections of HEK293T cells, whereas other results for HEK293T cells represent six independent experiments. All zebrafish-related assays were performed in three independent experiments and tested for comparison of two groups with unpaired one-tailed Student's t-test as follows:

[0181] result A first phase of WES analysis of known lymphatic abnormality-associated genes was unrevealing, including mutation analysis of AKT1, PIK3CA, KRAS, HRAS, NRAS, BRAF, RAF1, PTPN11, SHOC2, CBL, RIT1, and SOS1. Subsequent gene prioritization revealed a novel mutation, c.640T>C:p.S214P, in ARAF on chromosome X. Patient P1 is a male with CCLA (Fig. 22A, 22C-22E, see Methods for clinical details, GenBank Accession No. NG_016339), and patient P2 is a female with a previous diagnosis of lymphangiomatosis. The mutation affects a conserved phosphorylation site, likely resulting in a gain-of-function (GoF) effect, as residue 214 is a paralogous regulatory site in the homologous protein C-RAF (also known as RAF1) for inhibition by 14-3-3 proteins. This missense mutation was not present in the 1000 Genomes Project, ESP6500SI, ExAC v0.3, gnomAD v2.1, or in additional exome sequencing data from over 5,000 samples in our in-house database. Sanger sequencing of blood-derived DNA from P1 and both parents confirmed that this X-linked ARAF mutation occurred in somatic heterozygosity, as in the affected male (Fig. 22F). Sanger sequencing of the ARAF mutation in P2 and his unaffected daughter and mother confirmed that the mutation was present only in P2 (Fig. 22F). The father was unavailable for sequencing, but the absence of reported respiratory symptoms in the father suggests that the ARAF mutation in P2 likely arose as a de novo or somatic mutation. Patient P2 was lost to follow-up and was later informed that he died from complications of lymphatic disease 5 years after diagnosis.

[0182] One of the 14-3-3 binding sites of ARAF, Ser 214, is located in conserved region 2 (CR2) (14) and is highly conserved across vertebrate species, similar to RAF proteins, and is therefore thought to play an essential role in the function of these kinases (Figure 22G). Binding of 14-3-3 proteins to the phosphorylated 214th residue of ARAF is thought to prevent the recruitment of ARAF proteins to the cell membrane by activated Ras (15). Previous studies have shown that a mutation in the similar residue 259 of ARAF-S214 in C-RAF inhibits binding with 14-3-3 proteins, localizes them to the cell membrane, and induces ERK / MEK signaling (16). As shown in Figure 23A, ARAF-S214P-transfected HEK293T cells showed reduced co-precipitation of 14-3-3 proteins and significantly greater activation of ERK1 / 2, as measured by increased phosphorylation, compared with HEK293T cells expressing wild-type (WT) ARAF (Fig. 23A, 23B). Phosphorylation of AKT, p70S6K, mTOR, and p38 (another family of MAP kinases) was not altered by ARAF-S214P (Fig. 23B). Similar results were obtained in HeLa cells and primary human dermal lymphatic endothelial cells (HDLECs) (Fig. 23C). This marked hyperactivation was observed even in the absence of cytokines or growth factors.

[0183] HDLECs expressing ARAF-S214P had improved lymphangiogenesis compared to HDLECs expressing ARAF-WT, as measured by the number and length of sprouts in a 3D lymphoid spheroid sprouting assay performed in the absence of vascular endothelial growth factor C (VEGF) (Fig. 23D). The MEK inhibitor trametinib restored the increased sprouting of the mutant (Fig. 23D). We next performed a morphological analysis of endothelial adrenergic junctions in primary HDLECs expressing ARAF-S214P. Immunofluorescence microscopy revealed that expression of ARAF-S214P significantly abolished the accumulation of VE-cadherin between adjacent cells, suggesting enhanced internalization of VE-cadherin (Fig. 23E, yellow arrowheads). Furthermore, expression of ARAF-S214P altered the structure of actin, with the number of F-actin filaments in the cell body reduced in cells expressing the mutant.

[0184] We next examined the ability of MEK1 / 2 inhibitors to reverse these abnormalities. Treatment with the MEK inhibitor trametinib at a concentration of 100 nM restored the loss of VE-cadherin from cell-cell junctions observed in HDLECs expressing ARAF-S214P, almost completely restored cell monolayering, and restored normal appearance of VE-cadherin at junctions and actin filaments (Figure 23E). Although ARAF-S214P clearly activates ERK in HDLECs, and ERK activation is generally associated with cell proliferation, we did not observe any measurable difference in proliferation between cells expressing ARAF-WT and -S214P. HDLECs were transduced with two independent retroviral vectors (Figure 23F).

[0185] Analysis of lymphatic vessel development in zebrafish was performed in the Tg(mrc1a:egfp)y251 transgenic line. In this transgenic line, all lymphatic endothelial cells are labeled with EGFP. Expression of ARAF was targeted to lymphatic vessels using the mrc1a promoter, and ARAF-expressing cells were indicated by expression of mCherry. Expression of ARAF-S214P induced lymphatic vessel expansion in various locations, and most consistently, we observed expansion of trunk TDs (Figure 23G). On the other hand, expression of ARAF-WT also affected lymphatic vessel morphology (Figure 23H). Expression of ARAF-S214P induces p-ERK in zebrafish. To examine whether a MEK signaling inhibitor can restore the defect, mrc1a:ARAFS214P larvae were treated with cobimetinib starting from 3 days postfertilization (dpf), the time when lymphoid progenitor cells bud off and form TDs (17). Analysis of somites for ARAF expression revealed that cobimetinib significantly rescued duct morphology (Figures 23I, 23J). Meanwhile, administration of cobimetinib to WT Tg(mrc1a:egfp)y251 larvae revealed that the drug was well tolerated.

[0186] Given that we have shown that ARAF mutations lead to a gain of function in P1 that is insensitive to sirolimus and that MEK inhibitors rescue the lymphatic phenotype in endothelial cells and transgenic zebrafish models, we propose the following.

[0187] Permission was obtained from the Institutional Review Board to use MEK inhibitor treatment in P1, after which trametinib (Mekinist), a US Food and Drug Administration (FDA)-approved MEK inhibitor, was used off-label in this 12-year-old patient after a comprehensive baseline evaluation.

[0188] The starting dose of trametinib was 1 mg d -1Pulmonary function tests began to show improvement within 2 months of starting treatment (Figure 24A). Furthermore, 3 months after starting treatment, lymphatic fluid retention and supplemental oxygen requirements were significantly reduced, physical activity levels improved, and the patient was able to transition to room air without any trametinib-related adverse events. Pulmonary function tests 12 months after starting treatment showed that total lung capacity (TLC) had nearly doubled and forced expiratory volume in 1 second (FEV1) had improved from 23% to 42% of predicted (Figure 24A). Electrolytes (low Na, low K) normalized, and magnetic resonance imaging scans showed remodeling of the lymphatic system (Figures 24B-24F), with notable recovery including a doubling of total lung capacity (TLC) and forced expiratory volume (FEV1) (Figure 24C). FEV1 had improved from 23% to 42% of predicted (Figure 24A). Electrolytes (low Na, low K) normalized, and magnetic resonance imaging scans confirmed reconstitution of the lymphatic system (Figures 24B-24F). The patient, who had been frequently hospitalized before initiating this gene induction therapy, made a remarkable recovery (Figure 24F).

[0189] Thus, we performed WES in two unrelated patients with lymphatic abnormalities and identified recurrent gain-of-function mutations in the ARAF gene, including a 12-year-old male with advanced lymphatic disease unresponsive to sirolimus therapy. Introduction of mutant ARAF in HDLECs rescued the increased ERK1 / 2 activity, enhanced lymphangiogenesis, and disruption of the actin cytoskeleton and VE-cadherin junctions.

[0190] We performed this study using the MEK inhibitor trametinib. In HDLECs expressing ARAF-S214P, sprouting was observed in the absence of VEGFC (a potent lymphangiogenic factor) (18). In the same conditions, no sprouting was observed in cells expressing ARAF-WT. This suggests that ARAF mutants mimic the stimulatory behavior of VEGFC or that HDLECs induce the expression of VEGFC, which is necessary for endothelial cell sprouting as seen in many stromal cells (19-21). We recapitulated the abnormal lymphatic phenotype. This was done using a zebrafish model of a patient with a GoFARAF mutation, and observed phenotypic recovery by MEK inhibitors. Surprisingly, when trametinib was administered to a proband patient with an ARAF gene mutation, the patient's symptoms dramatically improved, and within 12 months of starting treatment, the dilated and tortuous lymphatic vessels were reconstructed, lymphedema was resolved, and the patient was able to resume normal daily activities.

[0191] In ongoing patient recruitment, we have investigated patients with more lymphatic anomalies, including Noonan (or Noonan-associated) syndrome, Gorham-Stout disease, scapular lymphangiomatosis (KLA), lymphangiectasia, and CCLA. We sequenced 43 more patients and identified seven additional mutations in KRAS, BRAF, RASA1, PTPN11, and SOS1 (Table 3). This suggests that RAS-MAPK signaling is a common pathway that drives the clinical manifestations of various lymphatic diseases. Indeed, it is increasingly recognized that the RAS-MAPK pathway plays an important role in signaling lymphangiogenesis (21-23). ​​We identified more than 50 patients with mutations in KRAS, HRAS, BRAF, RAF1, PTPN11, SHOC2, CBL, RIT1, and SOS1 and clinical features of Noonan syndrome or Noonan-associated syndrome with lymphatic anomalies, including pleural effusion, pericardial effusion, pneumothorax, and edema. During the course of our studies, recurrent NRAS mutations were identified in GLA (37) and KLA (38), further supporting a common genetic etiology for these disease groups and the importance of RAS-MAPK pathway mutations in lymphoid abnormalities.

[0192] The widespread presence of RASopathies mutations in human cancers has been recognized for decades. When we scrutinized the ARAF mutations we found using the cBioPortal (39) database (n=71,857 subjects, queried on February 6, 2019), we found two patients with the exact same mutation in ARAF. Interestingly, both patients also had TP53 mutations, which are considered to be oncogenic drivers. In addition, three of the different mutations at this residue (S214T, S214A, S214Y, S214C, S214F), which have been shown to increase MEK / ERK phosphorylation (40), were observed in 10 patients with different types of cancer. However, 9 out of 10 patients had coexisting oncogenic mutations in TP53, GNAS, AKT2, APC, EGFR, ATM, CHEK2, KIT, or U2AF1, and these oncogenic drivers may be responsible for the excessive proliferation of cancer cells. Although primary screeners with ARAF mutations have dilated lymphatic vessels, the size of the lesions has not increased over many years of follow-up. Thus, these data are consistent with our observation that ARAF mutations may not promote lymphatic vessel proliferation.

[0193] Endothelial cells in vitro Regarding the prevalence of mutation-positive lymphatic anomalies, which include but are not limited to GLA, Gorham-Stout disease, CCLA, KLA, Klippel-Trenaunay syndrome, kaposiform hemangioendothelioma, etc., among the 11 US centers forming the Lymphatic Anomalies Consortium to facilitate multicenter clinical trials of this group of lymphatic anomalies, there have been more than 3,000 patients recruited with moderate to severe disease course, with a combined annual number of new patients of approximately 300. Based on the current yield of molecular diagnostics (20%), we expect that approximately 20% of these will have RAS-MAPK pathway defects, suggesting the following: There could be thousands of patients across the US who would benefit from treatment with MEK inhibitors. Thus, our study illustrates how, with previously unknown etiological abnormalities in patients and the realization of precision medicine approaches, gene discovery can impact disease classification and uncover new biological and life-saving therapies.

[0194] literature 1. Trenor, CC3rd & Chaudry, G. Complex lymphatic anomalies. Semin. Pediatr. Surg. 23, 186-190 (2014). 2.Collins,FS&Varmus,HA new initiative on precision medicine.N.Engl.J.Med.372,793-795(2015). 3.Adams,DMet al.Efficacy and safety of sirolimus in the treatment of complicated vascular anomalies.Pediatrics 137,e20153257(2016). 4.Hammill,A.M.et al.Sirolimus for the treatment of complicated vascular anomalies in children.Pediatr.Blood Cancer 57,1018-1024(2011). 5.McCormick,A.,Rosenberg,S.,Trier,K.&Balest,A.A case of a central conducting lymphatic anomaly responsive to sirolimus.Pediatrics 137,e20152694(2016). 6.Hilliard,R.I.,McKendry,J.B.&Phillips,M.J.Congenital abnormalities of the lymphatic system:a new clinical classification.Pediatrics 86,988-994(1990). 7.Levine,C.Primary disorders of the lymphatic vessels-a unified concept.J.Pediatr.Surg.24,233-240(1989). 8.Smeltzer,D.M.,Stickler,G.B.&Fleming,R.E.Primary lymphatic dysplasia in children:chylothorax,chylous ascites,and generalized lymphatic dysplasia.Eur.J.Pediatr.145,286-292(1986). 9.Wassef,M.et al.Vascular anomalies classification:recommendations from the International Society for the Study of Vascular Anomalies.Pediatrics 136,e203-e214(2015). 10.Chen,W.,Adams,D.,Patel,M.,Gupta,A.&Dasgupta,R.Generalized lymphatic malformation with chylothorax:long-term management of a highly morbid condition in a pediatric patient.J.Pediatr.Surg.48,e9-e12(2013). 11.Lala,S.et al.Gorham-Stout disease and generalized lymphatic anomaly-clinical,radiologic,and histologic differentiation.Skeletal Radiol.42,917-924(2013). 12.Clemens,R.K.,Pfammatter,T.,Meier,T.O.,Alomari,A.I.&Amann-Vesti,B.R.Combined and complex vascular malformations.Vasa 44,92-105(2015). 13.Li,D.et al.Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly.Hum.Mol.Genet.27,3233-3245(2018). 14.Wellbrock,C.,Karasarides,M.&Marais,R.The RAF proteins take centre stage.Nat.Rev.Mol.Cell Biol.5,875-885(2004). 15.Lavoie,H.&Therrien,M.Regulation of RAF protein kinases in ERK signalling.Nat.Rev. Mol.Cell Biol.16,281-298(2015). 16.Molzan,M.et al.Impaired binding of 14-3-3 to C-RAF in Noonan syndrome suggests new approaches in diseases with increased Ras signaling.Mol.Cell Biol.30,4698-4711(2010). 17.Jung,H.M.et al.Development of the larval lymphatic system in zebrafish.Development 144,2070-2081(2017). 18.Karkkainen,M.J.et al.Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.Nat.Immunol.5,74-80(2004). 19.Carmeliet,P.&Jain,R.K.Molecular mechanisms and clinical applications of angiogenesis.Nature 473,298-307(2011). 20.Karaman,S.,Leppanen,V.M.&Alitalo,K.Vascular endothelial growth factor signaling in development and disease.Development 145,dev151019(2018). 21.Potente,M.&Makinen,T.Vascular heterogeneity and specialization in development and disease.Nat.Rev.Mol.Cell Biol.18,477-494(2017). 22.Coso,S.,Bovay,E.&Petrova,T.V.Pressing the right buttons:signaling in lymphangiogenesis.Blood 123,2614-2624(2014). 23.Brouillard,P.,Boon,L.&Vikkula,M.Genetics of lymphatic anomalies.J.Clin.Invest.124,898-904(2014). 24.Bulow,L.et al.Hydrops,fetal pleural effusions and chylothorax in three patients with CBL mutations.Am.J.Med.Genet.A 167A,394-399(2015). 25.Gargano,G.et al.Hydrops fetalis in a preterm newborn heterozygous for the c.4A>G SHOC2 mutation.Am.J.Med.Genet.A 164A,1015-1020(2014). 26.Gos,M.et al.Contribution of RIT1 mutations to the pathogenesis of Noonan syndrome:four new cases and further evidence of heterogeneity.Am.J.Med.Genet.A 164A,2310-2316(2014). 27.Hanson,H.L.et al.Germline CBL mutation associated with a Noonan-like syndrome with primary lymphedema and teratoma associated with acquired uniparental isodisomy of chromosome 11q23.Am.J.Med.Genet.A 164A,1003-1009(2014). 28. Milosavljevic,D.et al.Two cases of RIT1 associated Noonan syndrome: further delineation of the clinical phenotype and review of the literature.Am.J.Med.Genet.A 170,1874-1880(2016). 29.Koenighofer,M.et al.Mutations in RIT1 cause Noonan syndrome-additional functional evidence and expanding the clinical phenotype.Clin.Genet.89,359-366(2016). 30.Lee,K.A.et al.PTPN11 analysis for the prenatal diagnosis of Noonan syndrome in fetuses with abnormal ultrasound findings.Clin.Genet.75,190-194(2009). 31.Croonen,E.A.et al.Prenatal diagnostic testing of the Noonan syndrome genes in fetuses with abnormal ultrasound findings.Eur.J.Hum.Genet.21,936-942(2013). 32.Joyce, S. et al.The lymphatic phenotype in Noonan and cardiofaciocutaneous syndrome.Eur.J.Hum.Genet.24, 690-696 (2016). 33.Yaoita,M.et al.Spectrum of mutations and genotype-phenotype analysis in Noonan syndrome patients with RIT1 mutations.Hum.Genet.135,209-222(2016). 34.Lo,I.F.et al.Severe neonatal manifestations of Costello syndrome.J.Med.Genet.45,167-171(2008). 35.Ebrahimi-Fakhari,D.et al.Congenital chylothorax as the initial presentation of PTPN11-associated Noonan syndrome.J.Pediatr.185,248-248.e1(2017). 36. Morcaldi,G.et al.Lymphodysplasia and Kras mutation:a case report and literature review. Lymphology 48,121-127 (2015). 37.Manevitz-Mendelson,E.et al.Somatic NRAS mutation in patient with generalized lymphatic anomaly.Angiogenesis 21,287-298(2018). 38.Barclay S.F.et al.A somatic activating NRAS variant associated with kaposiform lymphangiomatosis.Genet.Med.https: / / doi.org / 10.1038 / s41436-018-0390-0(2018). 39.Gao, J. et al.Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.Sci.Signal.6,pl1(2013). 40.Imielinski,M.et al.Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma.J.Clin.Invest.124,1582-1586(2014).

[0195] Although some of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the present invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

1. 1. Use of one or more agents in the manufacture of a medicament for treating a lymphatic disorder in a human patient, comprising: (a) a single nucleotide variant (SNV) in one or more of KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, NF1, and CBL is detected in nucleic acid of a biological sample obtained from the patient; and (b) the drugs are each independently selected from the group consisting of trametinib (GSK1120212), rapamycin (sirolimus), everolimus (RAD001), AZD8055, temsirolimus (CCI-779, NSC 683864), KU-0063794, MHY1485, BEZ235 (NVP-BEZ235, dactolisib), PI-103, tolquinib (PP242), tacrolimus (FK506), selumetinib (AZD6244), PD0325901, PD184352 (CI-1040), pimasertib (AS-703026), TAK-733, AZD8330, binimetinib (MEK162, ARRY-162, ARRY-438162), SL-327, rifametinib (RDEA119, Bay86-9766), cobimetinib (GDC-0973, RG7420), ulixertinib, ridaforolimus (deforolimus, MK-8669), INK 128 (MLN0128), voxatalisib (SAR245409, XL765), torin1, omipalisib (GSK2126458, GSK458), OSI-027, PF-04691502, apitolisib (GDC-0980, RG7422), GSK1059615, jedatolicib (PF-05212384, PKI-587), WYE-354, AZD2014, torin2, WYE-125132 (WYE-132), P selected from the group consisting of P121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388, zotarolimus (ABT-578), tacrolimus (FK506), BGT226 (NVP-BGT226), Palomid 529 (P529), chrysophanic acid, TAK-733, PD-325901, pimasertib (AS-703026), PD184352, and SL-327; use.

2. In the use according to claim 1, the SNV is at least one (a) c.35G>A:pG12D of KRAS; (b) c.1403T>C:pF468S and c.2128-G>T of BRAF; (c) c.2536G>A:pE846K of SOS1; (d) ITGA9 c.1236+4A>G and c.289T>G: p.C97G; (e) RASA1 c.475_476del:p.(L159Gfs*20) and c.2246G>Cp.R749P; (f) c.433A>C:p.T145P of RAF1; (g) c.270G>T:p.M90I in RIT1; (h) c.7289C>T:p.P2430L of PIEZ01; (i) c.1034_1043del:p.(L345Pfs*28) of NF1, and (j) c.1096-1G>T and c.2322T>G of CBL: p.Y774*; Selected from, use.

3. The use of claim 1 or 2, wherein the lymphatic system abnormality is characterized by abnormal formation of lymphatic vessels and / or tissue overgrowth.

4. The use according to any one of claims 1 to 3, wherein the lymphatic system abnormality is lymphangiomatosis (LAM).

5. The use according to any one of claims 1 to 4, wherein the lymphatic system abnormality is generalized lymphatic abnormality (GLA).

6. The use according to any one of claims 1 to 5, wherein the lymphatic abnormality is characterized by chylous exudate, including pericardial, pleural, or peritoneal fluid.

7. The use of claim 1, wherein a report identifying the SNV is generated following detection in the biological sample.

8. The use of any one of claims 1 to 7, wherein a report is generated identifying a recommended treatment for the lymphatic system abnormality.

9. The use according to any one of claims 1 to 8, wherein the drug is at least one selected from the group consisting of trametinib, pimasertib, and rifametinib.

10. The use of any one of claims 1 to 9, wherein the patient does not have one SNV selected from the group consisting of PTPN11, KRAS, BRAF, SOS1, and ITGA9.

11. The use of claim 1, wherein (a) further comprises analyzing the polynucleotide sample to determine the presence of the SNV by performing a process selected from the group consisting of detection of specific hybridization, measurement of allele size, restriction fragment length polymorphism analysis, allele-specific hybridization analysis, single-base primer extension reaction, and sequencing of the amplified polynucleotide.

12. The use according to any one of claims 1 to 11, wherein the biological sample comprises DNA or RNA.

13. The use according to any one of claims 1 to 12, wherein the nucleic acid containing the SNV is obtained from an isolated cell of a human patient.

Images