Compositions and methods for the diagnosis and treatment of lymphatic system diseases

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 on 7 September 2018, which is incorporated herein by reference as if its entire contents were fully described therein.

[0002] This invention relates to the fields of genetics, personalized medicine, and lymphoid malformations. Specifically, it provides novel gene targets and therapeutic regimens for improving symptoms associated with lymphangiomatosis and other generalized lymphoid malformations (GLA). [Background technology]

[0003] This specification incorporates references to several publications and patent documents to illustrate the state of the art related to the present invention. These references are incorporated herein by reference as if they were all contained herein.

[0004] The lymphatic system plays a vital role in maintaining fluid circulation, protecting the body from disease, and absorbing dietary fats in the small intestine (1). Complex lymphatic disorders are characterized by abnormal formation of lymphatic vessels and excessive tissue proliferation. Patients often present with overlapping symptoms and may develop severe lung disease (2, 3). Examples of lymphatic disorders include generalized lymphangiopathy (GLA), lymphangiectasia, and biliary retention (pericardial, pleural, peritoneal). Research into complex lymphatic disorders is hampered by inconsistent classification and nomenclature, which poses significant diagnostic challenges (3-6). The molecular genetic etiology of complex lymphatic disorders is not well understood, but there appears to be a related genetic etiology to congenital lymphatic malformations (7-9). In fact, both germline and somatic mutations have been identified in genes concentrated 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 dilation and remodeling in the construction of mature lymphatic networks, and such disruptions are associated with lymphatic disorders. Acquisition of functional mutations in AKT1 and PIK3CA results in elevated mammalian target of rapamycin complex 1 (mTORC1) activity and has been observed in patients with lymphatic malformations that constitute some 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 disrupt the regulation of RAS pathway activity, leading to lymphedema and lymphangiectasia in conditions 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 disorders and abnormalities such as lymphangiomatosis / lymphangiectasia (LAM), generalized lymphatic malformation (GLA), and pleural effusion, as well as therapies that target genetic markers associated with these diseases. The following are prior art documents related to the invention of this application (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries): (Prior art document) (Patent Document) (Patent Document 1) International Publication No. 2018 / 045078 (Patent Document 2) International Publication No. 2014 / 186750 [Overview of the project] [Means for solving the problem]

[0007] Accordingly, one embodiment of the present invention provides a method for diagnosing lymphatic abnormalities in a human patient. An exemplary method includes the steps of: obtaining a biological sample containing nucleic acids from a patient; assaying the nucleic acids to determine whether i) one or more single nucleotide variants (SNVs) of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF are present, or ii) one or more SNVs of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF are in linkage disequilibrium; and diagnosing the patient with a lymphatic abnormality if the SNVs of i) or ii) are present. In another embodiment, a method for diagnosing lymphatic abnormalities in a human patient, comprising the steps of: obtaining genotype sequence information from a human patient; determining from the sequence information whether i) one or more single nucleotide variants (SNVs) of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF are present, or ii) one or more SNVs of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, CBL, and ARAF are in linkage disequilibrium; and diagnosing the patient with a lymphatic abnormality if the SNVs of i) or ii) are present.

[0008] The present invention also provides a method for treating lymphatic abnormalities in human patients. An exemplary method includes the steps of: obtaining a biological sample containing nucleic acids from a patient; assaying the nucleic acids to determine whether i) one or more single nucleotide variants (SANVs) from among PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL are present, or ii) one or more SNVs from among PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL are present in linkage disequilibrium with an SNV; and administering one or more agents suitable for treating the lymphatic abnormality to a patient identified as having one or more SNVs from i) or ii), thereby treating the lymphatic abnormality. An alternative embodiment of this method includes the steps of: obtaining genotype information from a patient; performing an assay to determine whether i) one or more single nucleotide variants (SNVs) from among PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL are present, or ii) one or more SNVs from among PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, and CBL are in linkage disequilibrium; and administering one or more drugs suitable for treating the lymphatic abnormality to patients identified as having one or more SNVs from i) or ii), thereby treating the lymphatic abnormality. In the alternative embodiment of this method, genotype information is obtained from the 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 yet another embodiment, the lymphatic abnormality is generalized lymphatic malformation (GLA). The lymphatic abnormality may be characterized by chylous effusions, including pericardial fluid, pleural fluid, or peritoneal fluid.

[0010] The diagnostic method may further include a step of generating a report that identifies SNVs after detection in a biological sample. The treatment method described above may further include a step of generating a report that identifies recommended treatment(s) for the lymphatic abnormality based on the SNVs identified by the said method.

[0011] In some embodiments, the drugs administered to subjects with SNV-positive status are: 1) MEK / ERK inhibitors; 2) drugs / inhibitors listed in Tables 1 and 2; 3) combinations of MEK / ERK inhibitors and one or more drugs / inhibitors listed in Tables 1 and 2; and / or 4) combinations of 1) mTOR inhibitors and / or PIK3K inhibitors, and 2) one or more MEK / ERK inhibitors. In yet another embodiment, the diagnostic method described herein may further include the step of administering an effective dose of one or more drugs suitable for treating the lymphatic abnormality to the diagnosed patient.

[0012] In certain embodiments of the treatment method, the drug administered to patients with one or more lymphoma-associated SNVs is selected from one or more MEK / ERK inhibitors and one or more combinations of said inhibitors. In some embodiments, the drug administered is a MEK / ERK inhibitor. In some embodiments, the drug administered is one or more drugs listed in Tables 1 and 2. In some embodiments, the drug administered is a MEK / ERK inhibitor and one or more drugs listed in Tables 1 and 2. In some embodiments, the drug administered is 1) an mTOR inhibitor and / or a PIK3K inhibitor; and 2) a combination of one or more MEK / ERK inhibitors. In some embodiments, if the drug is an mTOR inhibitor, rapamycin and / or BEZ-235 (dactricib) are administered. In certain embodiments, one or more mTOR inhibitors, one or more PIK3K inhibitors, and / or 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 drugs listed in Tables 1 and 2 are used in combination. These combinations include, but are not limited to, a) ridafololimus and trametinib, b) ridafololimus and selumetinib or cobimetinib, c) BEZ235 and selumetinib, d) omiparisib and selumetinib or trametinib, e) everolimus and trametinib or selumetinib, f) sirolimus, ridafololimus and selumetinib, g) sirolimus, ridafololimus and trametinib, h) tolkinib and trametinib, i) BEZ235, tolkinib and trametinib, and j) sirolimus, gedatricib and trametinib. In other embodiments, the treatment further includes administering systemic chemotherapy, interferon alpha, radiotherapy, and / or surgery.

[0015] In some embodiments, SNVs are selected from SNVs selected from the following: 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 and c.289T>G:p.C97G of the ITGA9 gene, where "c." indicates an encoded DNA sequence and "p." indicates a protein sequence.

[0016] In some embodiments, the diagnostic method includes the detection of one or more of the SNVs described above. In some embodiments, the diagnostic method further includes the step of administering one or more agents known to treat lymphatic abnormalities to a subject. In some embodiments, the agent 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. Administration of the agent improves one or more of the following: improvement of lymphatic structure, reduction of chylothorax, improvement of respiratory function, enabling gradual reduction of concomitant medication use, and / or an increase in survival rate.

[0017] In some embodiments, the diagnostic method includes the steps of 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, either alone or in combination. In other embodiments, the drugs are selected from Tables 1-2, thereby achieving one or more of the following: improvement of lymphatic structure, reduction of bile in pleural effusion, improvement of respiratory function, gradual reduction of the dosage of concomitant drugs, or improvement of survival rate.

[0018] In some embodiments, the diagnostic method includes the detection of c.1403T>C:pF468S in the BRAF gene. In some embodiments, the diagnostic method further includes the step of treating the patient with one or more MEK / ERK inhibitors alone or in combination. In other embodiments, the drugs are selected from Tables 1-2, thereby improving one or more of the following: improvement of lymphatic structure, reduction of pleural effusion ethylation, improvement of respiratory function, gradual reduction of the dosage of concomitant drugs, or increased survival rate.

[0019] In some embodiments, the diagnostic method includes detecting c.35G>A:pG12D of 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 is selected from Tables 1-2 and administered, thereby improving one or more of the lymphatic structures, reducing pleural effusion, improving respiratory function, allowing for a gradual reduction in the amount of concomitant medication used, or improving survival rate.

[0020] In some embodiments, the diagnostic method includes detecting c.2536G>A:pE846K in the SOS1 gene. In some embodiments, the diagnostic method further includes treating the patient with one or more MEK / ERK inhibitors alone or in combination. In other embodiments, an agent from Tables 1-2 is selected, thereby improving one or more of the improvement of lymphatic structure, reduction of bile in pleural effusion, improvement of respiratory function, allowing for a gradual reduction in the amount of concomitant medication used, or increase in survival rate.

[0021] In some embodiments, the diagnostic method includes detecting c.1236+4A>G and / or c.289T>G:p.C97G of 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, thereby improving one or more of the lymphatic structures, reducing bile in pleural effusion, improving respiratory function, allowing for a gradual reduction in concomitant medication use, or increasing survival rate.

[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, to determine whether there is a single nucleotide variant (SNV) in one or more of 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, c.1403T>C:pF468S of the BRAF gene, c.2536G>A:pE846K of the SOS1 gene, and c.1236+4A>G and c.289T>G:p.C97 of the ITGA9 gene, the step of assaying for proliferation further includes 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.

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

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

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

[0027] In some embodiments, the isolated vector encodes a nucleic acid having an SNV, and the SNV is 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, and c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene.

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

[0029] In some embodiments, the method for screening the effects of a drug includes contacting host cells or transgenic animals with one or more inhibitors described herein, either alone or in combination, but also encompassing the drugs listed in Tables 1-2. In some embodiments, the effect of the drug being screened is tail rescue or branch rescue in zebrafish.

[0030] In some embodiments, a method for identifying a drug that alters cellular signaling includes the steps of: providing cells expressing a nucleic acid containing at least one SNV as described above and cells expressing a congeneral wild-type sequence lacking the SNV; contacting both cell populations with a drug of test; and analyzing whether the drug alters the cellular signaling of the cells possessing the nucleic acid containing the SNV compared to the cells lacking the SNV. [Brief explanation of the drawing]

[0031] [Figure 1] Figure 1. Overexpression of BRAF and PTPN11 mutants in the Ea.hy926 cell line activates the ERK signaling pathway. [Figure 2]Figure 2. Overexpression of BRAF F486S in the Ea.hy926 cell line alters cell morphology and actin structure. [Figure 3] Figure 3. Administration of the MEK inhibitor trametinib increases VE-cadherin surface staining and filamentous actin in cells overexpressing BRAF WT and F486S mutants. [Figure 4A] Figures 4A-4B. MEK inhibitors reduce ERK activation / phosphorylation in cells overexpressing BRAFWT and F486S mutants. [Figure 4B] Figure 4B-MEK inhibitors reduce ERK activation / phosphorylation in cells overexpressing BRAFWT and F486S mutants. [Figure 5A] Figures 5A-5B. MEK inhibitors suppress ERK activation / phosphorylation in cells overexpressing the PTPN11 mutant (Figure 5A). [Figure 5B] MEK inhibitors suppress ERK activation / phosphorylation in cells overexpressing PTPN11 mutants (Figure 5B). [Figure 6] Figure 6. Effect of PD0325901 on HDLEC expressing ARAF mutant S214P. Cell morphological differences induced by ARAF-S214P were rescued by PD0325901, indicated by decreased pERK levels and increased accumulation of VE-cadherin at the cell membrane. Green indicates HA staining marking ARAF-expressing cells, and red indicates VE-cadherin staining. [Figure 7] Figure 7. Effect of CI1040 on HDLEC expressing ARAF mutant S214P. Cell morphological differences induced by ARAF-S214P were restored by CI1040, as indicated by decreased pERK levels and increased accumulation of VE-cadherin in the cell membrane. Green indicates HA staining marking ARAF-expressing cells, and red indicates VE-cadherin staining. [Figure 8]Figure 8. Effect of Pimasertib on HDLEC expressing ARAF mutation S214P. Cell morphological differences induced by ARAF-S214P were rescued by pimasertib, as indicated by decreased pERK levels and increased accumulation of VE-cadherin in the cell membrane. Green indicates HA staining marking ARAF-expressing cells, and red indicates VE-cadherin staining. [Figure 9] Figure 9. Effect of TAK-733 on HDLEC expressing ARAF mutation S214P. Cell morphological differences induced by ARAF-S214P were rescued by TAK-733, indicated by decreased pERK levels and increased accumulation of VE-cadherin in the cell membrane. Green indicates HA staining marking ARAF-expressing cells, and red indicates VE-cadherin staining. [Figure 10] Figure 10. Effect of AZD8330 on HDLEC expressing ARAF mutant S214P. Cell morphological differences induced by ARAF-S214P were restored by AZD8330, as indicated by decreased pERK levels and increased accumulation of VE-cadherin in the cell membrane. Green indicates HA staining marking ARAF-expressing cells, and red indicates VE-cadherin staining. [Figure 11] Figure 11. Effect of rifametinib on HDLEC expressing ARAF mutation S214P. Cell morphological differences induced by ARAF-S214P were restored by rifametinib, as indicated by decreased pERK levels and increased accumulation of VE-cadherin in the cell membrane. Green indicates HA staining marking ARAF-expressing cells, and red indicates VE-cadherin staining. [Figure 12] Figure 12. Effect of urixertinib on HDLEC expressing ARAF mutation S214P. Cell morphological differences induced by ARAF-S214P were restored by urixertinib, as indicated by decreased phosphorylation levels of the ERK substrate RSK3 and increased VE-cadherin accumulation in the cell membrane. Green indicates HA staining showing ARAF-expressing cells, red indicates VE-cadherin staining, and white indicates DAPI staining showing the nucleus. [Figure 13]Figure 13. Effect of urixertinib on HDLEC expressing BRAF mutant F486S. Cell morphological differences induced by BRAF-F486S were restored by urixertinib, as indicated by increased VE-cadherin accumulation in the cell membrane. Green is HA staining marking BRAF-expressing cells, red is VE-cadherin staining, and white is DAPI staining marking the nucleus. [Figure 14] Figure 14. Effect of trametinib on HDLEC expressing BRAF mutant F486S. Cell morphological differences induced by BRAF-F486S were restored by trametinib, as indicated by decreased ERK phosphorylation levels and increased VE-cadherin accumulation in the cell membrane. Green is HA staining marking BRAF-expressing cells, red is VE-cadherin staining, and white is DAPI staining marking the nucleus. [Figure 15] Figure 15. Effect of trametinib on HDLEC expressing RAF1 mutant T145P. Cell morphological differences induced by RAF1-T145P were restored by trametinib, as indicated by decreased ERK phosphorylation levels and increased accumulation of VE-cadherin in the cell membrane. [Figure 16] Figure 16. Effects of urixertinib and trametinib on HDLECs expressing KRAS mutation G12D. Cell morphological differences induced by KRAS-G12D were restored by trametinib and urixertinib, as indicated by decreased ERK phosphorylation levels and increased accumulation of VE-cadherin in the cell membrane. [Figure 17] Figure 17. Weak activation of p-ERK induced by RIT1 mutation. [Figure 18] Figure 18. In the 3D lymphocyte spheroid sprouting assay, the sprouting activity of HDLECs expressing three different EPHB4 mutations was increased compared to EPHB4-WT, as measured by sprouting length in the lower panel. 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 resulted in significant improvement in edema induced by KRAS mutation G12D. [Figure 20] Figure 20. Mosaic expression of the S502A or G503R mutation in PTPN11 resulted in a phenotype of mild lymphatic malformation. [Figure 21] Figure 21. When residue 749 of rasa1a and rasa1b is knocked out using CRISPR, large edema forms in zebrafish, but no phenotype is obtained with single targeting (rasa1a or rasa1b) or ATG targeting. [Figure 22A-E] Figures 22A-22G. Clinical features and molecular analysis of lead probands of lymphatic abnormalities. Figure 22A) Coronal slice of T2-weighted non-contrast lymphangiography showing large pericardial effusion (arrow). Figure 22B) Maximum intensity projection of dynamic contrast-enhanced MRI lymphangiography of a healthy control subject, showing normal TD flowing toward the left internal vein bifida. Figure 22C) Maximum intensity projection of contrast-enhanced MRI lymphangiography of P1. Shows a dilated lumberian lymphatic network with retrograde hilar flow (arrowhead) and a dilated, tortuous TD flowing toward the left internal vein bifida (arrow), further supplying retrograde perfusion to the mediastinum and pericardium (box). Figure 22D) Contrast-enhanced lymphangiography of the box region of c. The distal part of the TD is dilated and tortuous, and it can be seen that there is retrograde flow toward the mediastinum, pericardium, and lungs via a dilated lymphatic network originating from the distal part of the TD (arrow). Figure 22E) Coronal maximum intensity projection of the pelvis and genitals. Multiple dilated canals (arrowheads) originating from bilateral inguinal lymph nodes are visible, supplying retrograde flow to the penis and scrotum (arrows). [Figure 22F] Figure 22F) Lineage and genotype of the recurrent ARAF mutation, c.640T>C(p.S214P), identified in unrelated families. [Figure 22G] Figure 22G) Schematic topology of the ARAF protein. This Ser214 residue is highly conserved in vertebrates and all RAF isoforms. [Figure 23A]Figures 23A-23J. The ARAF-S214P mutation increases ERK1 / 2 activity, enhances lymphangiogenesis, alters the actin cytoskeleton and VE-cadherin junctions in HDLECs, and leads to thoracic duct (TD) dilation in zebrafish, which is reversed by cobimetinib. Figure 23A), transfection of HEK293T cells with the ARAF mutant impairs binding to 14-3-3 protein and increases p-ERKs. The normalized 14-3-3 / FLAG ratio is shown in the right panel, showing that co-immunoprecipitation of 14-3-3 protein is reduced in the mutant. Data are shown as mean ± sem from three independent experiments. Two-sided, matching paired t-test (4 degrees of freedom), ****P = 8.6 × 10⁻⁶. Images have been cropped for better viewing. [Figure 23B] Figure 23B), transfection with the ARAF mutant in HEK293T cells induces increased p-ERK1 / 2 expression compared to cells expressing WT (**P=0.0026; bilateral unpaired t-test; df=8). Phosphorylation of AKT, p70S6K, mTOR, and p38 was not altered by ARAF-S214P. Normalized ratios are shown in the box plot on the right (showing all points from minimum to maximum), with the median line representing the median, the box limits representing the interquartile range, and the whiskers representing the data range from minimum to maximum. Six independent experiments were performed. Images have been cropped for better viewing. [Figure 23C-D]Figure 23C) Primary HDLEC cells transfected with ARAF-WT or ARAF-S214P were cultured in increasing concentrations of trametinib. Results from cells from three independent transformations were quantified and graphed in a scatter dot plot with each individual value superimposed as a dot. Data are shown as the mean ± sem (error bars) of the three independent experiments. Introduction of ARAF-S214P significantly increased p-ERKs levels (*P=0.03; two-tailed unpaired t-test with 4 df). Trametinib treatment resulted in a significant decrease in p-ERKs (*P=0.02 for 100nM trametinib treatment and 300nM trametinib treatment; *P=0.01 for 1,000nM trametinib treatment and 3,000nM trametinib treatment, two-tailed unpaired t-test with 4 df); NS, not significant. Images have been cropped for better viewing. Figure 23D) The three-dimensional lymphocyte spheroid sprouting assay shows increased sprouting activity in HDLEC expressing ARAF-S214P compared to ARAF-WT, measured by both sprouting number (***P=0.0002) and basal sprouting length (***P=0.0005). Two-tailed unpaired t-test with 22 df. Furthermore, when spheroids were cultured with increasing concentrations of trametinib, the number of shoots decreased 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 shoots decreased 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). Two-tailed opposed t-tests were used. Three experiments performed with independent introduction of HDLECs were quantified, and points from all three experiments were plotted on interleaved box plots (3-6 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 value of the data. [Figure 23E](Figure 23E) The ARAF mutant affected the localization of VE-cadherin (****P=1.88×10⁻²⁶), and treatment with trametinib increased the localization of VE-cadherin on the cell surface (****P=1.63×10⁻¹⁹). Red arrowheads indicate cell membrane staining, and yellow arrowheads indicate intracellular staining. In three experiments where HDLECs were introduced independently, intracellular and cell membrane staining was quantified, and the points from the three experiments were plotted in the box plot on the left (minimum to maximum) (75 points per condition). The central line represents the median, the box limits represent the interquartile range, and the whiskers represent the data range from minimum to maximum. A two-sided diagonal t-test of 148df, NS, is not significant. The maximum length and width of cells obtained in the experiment in Figure 23E were measured, and the ratio of length to width was calculated and plotted in the box plot on the right (minimum to maximum; bottom right). The central line represents the median, the box limits represent the interquartile range, and the whiskers represent the data range from the minimum to the maximum value. Expression of ARAF-S214P significantly increased the aspect ratio (****P=9.57×10⁻¹⁵), and treatment with trametinib normalized the aspect ratio (****P=7.51×10⁻¹⁷; 148df bilateral opposed t-test; NS, not significant). [Figure 23F] Figure 23F) The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay shows that ARAF-S214P does not result in increased proliferation in transfected HDLEC. Metabolic activity is measured at two wavelengths (550 nm and 700 nm). The contents of three wells (n=3 independent wells) were collected at the indicated times as described in "Methods". The trend line shows the mean value of each transderm at each time point, and the dots show the measured values ​​for all data points. This experiment is representative of the results obtained from the transduction of two independent retroviruses. [Figure 23G-J]Figures 23G-23I) Top: Overview of the fish lymphatic system. The white frame indicates the areas investigated in Figures 23G-23I, showing the confocal scan overlay (maximum intensity projection). In Figures 23G-23I, the TD and posterior central vein (PCV) are shown in green (gg(mrc1a:EGFP)), and the TD is shown with a dotted line. ARAF transgenic (mrc1a:araf) cells are shown in red (green + red → yellow). Figure 23G) Expression of ARAF-S214P causes severe dilation of the TD. Figure 23H) Expression of ARAF-WT (red) does not affect the morphology of the TD and PCV (both green). Figure 23I) Cobimetinib (1uM) partially reverses the mutation-induced dilation. Three independent experiments were repeated, yielding results similar to those in Figures 23G-23I. (Figure 23J) Phenotypic scoring categories for segmental dilation with and without cobimetinib treatment: normal, moderate dilation (TD is dilated but can be separated from PCV), severe dilation (TD and PCV overlap). Cobimetinib administration significantly suppressed severe dilation (****P=1.33×10⁻⁵; unilateral unpaired t-test; blue histogram) and restored normal morphology (***P=0.00051; unilateral unpaired t-test; green histogram). A total of 40 larvae and 120 segments were analyzed in three independent experiments. Data are presented as mean ± sem of three independent experiments. Untreated blot images are provided as source data. [Figure 24A] Figures 24A-24F show pulmonary function tests and clinical images of lead probands before and after treatment with MEK inhibitors. Figure 24A) Results of pulmonary function tests before and after treatment with MEK inhibitors. Note that all spirometry measurements significantly improved (marked in red): FEV1 increased from 23% to 42% of the predicted value, TLC increased from 29% to 56% of the predicted value, and Maximum Inspiratory Pressure (MIP) increased from 71% to 115% of the predicted value. FVC is forced survival capacity, FEF25-75 is intermediate forced expiratory flow rate, 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) Coronary maximum intensity projection images of T2-weighted non-contrast lymphatic vessels immediately before the start of medical treatment (left) and 12 months after the start of MEK inhibitor treatment (right) show that the extensively dilated and beaded subcutaneous lymphatic vessels have been almost completely absorbed. [Figure 24C] Figure 24C) Coronary maximum intensity projection of contrast-enhanced lymphangiography of the pelvis and chest before treatment (left) shows that there are few central lymphatic vessels, no central lymphatic flow above the diaphragm, and the bilateral subcutaneous canals running along the abdominal wall are greatly dilated and bead-like. Twelve months after the start of treatment (right), the dilated subcutaneous canals have been absorbed, and a new lymphatic network with a more normal appearance has formed, extending along the abdominal wall to the chest. [Figure 24D] Figure 24D) The coronal maximum intensity projection of contrast-enhanced lymphangiography of the pelvis and femur before treatment (left) shows that there are few ducts in the femur, and the lymphatic vessels are greatly dilated and beading. After treatment (right), the abnormally dilated ducts have been absorbed, and a new, more normal-looking lymphatic network has been formed. [Figure 24E] Figure 24E) Chest X-rays taken before treatment (left) and 12 months after treatment (right) show a reduction in pleural effusion and a significant improvement in lung capacity. [Figure 24F] Figure 24F) Patient growth record (left). Treatment with trametinib was started just before the age of 13 (*), and improvement in lymphedema and clinical symptoms was observed approximately 3 to 6 months after the start of treatment. The upper right image is a photograph of the patient's lower limbs immediately after removing compression stockings at peak weight. The lower right image is a photograph of the corresponding lower limbs. [Modes for carrying out the invention]

[0032] The Ras / mitogen-activated protein kinase (MAPK) pathway plays a crucial role in cell proliferation, migration, differentiation, and apoptosis, all of which are essential for normal development. Central conduit lymphangiopathy (CCLA) is a complex lymphangiopathy characterized by lymphatic dilation, lymphatic motility disorders, and distal occlusion of lymphatic vessels. CCLA was first reported by Trenor III, Chaudry, and Clemens et al., and in 2015 it was classified as a channel lymphangiomalformation by the International Society for Research on Vascular Anomalies (ISSHA). Clinical symptoms show a pattern that largely overlaps with generalized lymphangiopathy (GLA), a closely related diagnostic term, including chylothorax, hylous ascites, lymphatic leakage or reflux, and limb swelling. We recently identified gain-of-function mutations in ARAF as a cause of lymphatic abnormalities, including LAM, GLA, and CCLA. This section introduces gain-of-function mutations in KRAS, BIRAF, and PNPN11 as causative mutations of lymphoid abnormalities, including LAM, GLA, and CCLA. It also reveals that mutations in SOS1, ITAG9, RASA1, RAF1, RIT1, PIEZO1, EPHB4, NF1, CBL, and ARAF contribute to the development of this disease.

[0033] Lymphatic disorders are a rare and devastating disease spectrum, mostly of unknown cause, and treatment varies depending on the patient's symptoms. Identifying the causative genes will enable the development of inexpensive treatments aligned with precision medicine practices.

[0034] In Example II, a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) was characterized in a 12-year-old boy with a progressive abnormal lymphoid disease unresponsive to conventional sirolimus therapy and in another unrelated adult patient. This mutation resulted in the loss of a conserved phosphorylation site. Cells introduced with ARAF-S214P showed increased ERK1 / 2 activity, enhanced lymphangiogenesis, and disruption of the actin cytoskeleton and VE-cadherin junctions, which could be restored using the MEK inhibitor trametinib. Furthermore, the functional relevance of this mutation was verified by reproducing the lymphatic phenotype in a zebrafish model and rescuing the abnormal phenotype using a MEK inhibitor. Subsequently, treatment with the MEK inhibitor resulted in dramatic clinical improvements in the patients, including lymphatic system remodeling, resolution of lymphedema, significant improvement in pulmonary function tests, discontinuation of oxygen supplementation, and near-normalization of daily life. These results are a prime example of how understanding gene classification and mechanisms can guide evidence-based therapies, and in our case, it saved lives.

[0035] Next, specific embodiments of the present invention will be referred to in detail, examples of which are shown in the accompanying drawings. While the present invention will be described in relation to the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. Rather, the present invention is intended to cover all substitutes, modifications, and equivalents that may be included in the present invention as defined by the accompanying claims.

[0036] Before describing in detail the teachings of the present invention, it should be understood that this disclosure is not limited to any particular composition or process, and such may vary. It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to “a conjugate” includes multiple conjugates, a reference to “a cell” includes multiple cells, and so on.

[0037] It will be understood that there is an implicit "approximation" before the temperatures, concentrations, times, etc., discussed in this disclosure, such that minor and insubstantial deviations are within the scope of the teachings herein. Furthermore, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not intended to be limiting. It should be understood that the above general and detailed descriptions are illustrative and descriptive and do not limit the teachings.

[0038] Unless otherwise specified in the above specification, embodiments described in the specification as "comprising" of various components are also intended to "consist of" or "consisting essentially of" the components described. Furthermore, in this specification, embodiments described as "consisting essentially of" various components may also be considered to "consist of" or "comprising" the components described (this interchangeability does not apply to the use of these terms in the claims).

[0039] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. In the event of any conflict between references incorporated herein and any terms defined herein, this specification shall prevail. While the teachings of the present invention are described in conjunction with various embodiments, the teachings of the present invention are not intended to be limited to such embodiments. Rather, these teachings encompass a variety of alternatives, modifications, and equivalents, as will be understood by those skilled in the art.

[0040] Furthermore, the compounds “selected from the group consisting of ~” are one or more compounds listed below, and also include mixtures (i.e., combinations) of two or more compounds. According to the present invention, an isolated or biologically pure molecule is a compound removed from its natural environment. Thus, “isolated” and “biologically pure” do not necessarily reflect the degree to which the compound has been purified. The isolated compounds of the present invention can be obtained from their natural sources, can be produced using laboratory synthesis techniques, or can be produced by any such chemical synthesis route.

[0041] "Lymphatic abnormalities" refer to diseases or disorders characterized by abnormal formation of lymphatic vessels and overgrowth of tissues. Non-exclusive examples of lymphatic abnormalities include "lymphangiomatosis" or "lymphangiectasia" (collectively referred to as LAM in this specification), lymphangiomas, generalized lymphangioma (GLA), and chyle exudate, generalized lymphangiomas, generalized cystic hemangioma, multiple lymphangiectasia, generalized lymphangiomalformations, CCLA, diffuse lymphangiomalformations, Kaposiform LAM, and Gorham-Stout disease (GSD), a rare lymphoid vascular disorder characterized by progressive osteolysis.

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

[0043] A single nucleotide polymorphism (SNV) refers to the presence of a single base different from the usual bases at a specific position on genomic DNA. SNVs are similar to SNPs, but while SNPs generally refer to SNPs that occur with a certain frequency (for example, occurring in a certain percentage of the population), there is no frequency information for SNVs. Millions of SNVs are cataloged in the human genome. Some SNVs cause disease, while others are normal genomic mutations.

[0044] A "lymphatic disorder-related SNV or specific marker" is an SNV that is associated with an increased risk of developing a lymphatic disorder and is not found in patients who do not develop the disorder. Such markers include, but are not limited to, nucleic acids, the proteins encoded by them, or other small molecules.

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

[0046] Linkage refers to the tendency for genes, alleles, loci, or genetic markers to be inherited together due to their location on the same chromosome. This tendency is measured by the recombination rate (also called the recombination rate or θ) between two genes, alleles, loci, or genetic markers. The closer two loci on a chromosome are physically, the lower the recombination rate. Typically, when examining the association between a polymorphic region in a disease-causing gene and the disease itself, the recombination rate is 0, indicating that the disease and the disease-causing gene are always linked. Rarely, if a gene occupies a very large portion of the genome, recombination may be observed between a polymorphic region at one end of the gene and the causative mutation at the other end. However, if the causative mutation is the polymorphism being examined to investigate its association with the disease, no recombination is observed.

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

[0048] Linkage disequilibrium, or allele association, means that a particular allele, locus, gene, or genetic marker is preferentially associated with a particular allele, locus, gene, or genetic marker located in a nearby chromosomal position at a higher frequency than would be expected by chance for the frequency of that particular allele in a population.

[0049] The term "solid matrix" as used herein refers to any form, including beads, microparticles, microarrays, the surface of microtitration wells or test tubes, dipsticks, and filters. The matrix material may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran, or agarose. The solid matrix may contain nucleic acids immobilized on it so as not to be 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, wherein the defined wild-type region contains at least one known nucleotide mutation, which may or may not be associated with lymphatic abnormalities. Nucleic acid molecules may be isolated from natural sources by cDNA cloning or subtractive hybridization, or they may be synthesized manually. Nucleic acid molecules may be synthesized manually by triester synthesis, or they may be synthesized using an automated DNA synthesizer.

[0051] With respect to nucleic acids used in this invention, the term "isolated nucleic acid" as applied to DNA means a DNA molecule that has been separated from the immediately contiguous sequence (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. For example, "isolated nucleic acid" may consist of a DNA molecule inserted into a vector such as a plasmid or viral vector, or a DNA molecule incorporated into the genomic DNA of a prokaryote or eukaryote. Furthermore, "isolated nucleic acid molecule" may 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" primarily refers to RNA molecules encoded by the isolated DNA molecules defined above. The term can also refer to RNA molecules that are sufficiently separated from the RNA molecules they would naturally be associated with (i.e., within cells or tissues) and exist in a "substantially pure" form.

[0053] Using the term "enriched" in relation to nucleic acids means that a particular DNA or RNA sequence constitutes a significantly higher proportion (2-5 times) of the total DNA or RNA present in the cell or solution of interest compared to normal cells or cells from which the sequence was taken. This can be caused by a preferential decrease in the amount of other DNA or RNA present, a preferential increase in the amount of the particular DNA or RNA sequence, or a combination of both. However, it should be noted that "enriched" does not mean the absence of other DNA or RNA sequences, but merely that the relative amount of the sequence of interest has increased significantly.

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

[0055] The term "complementary" refers to two nucleotides that can form multiple beneficial 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. Therefore, if a nucleic acid sequence contains the base sequence thymine, adenine, guanine, and cytosine, the "complement" of this nucleic acid molecule will be a molecule containing adenine instead of thymine, thymine instead of adenine, cytosine instead of guanine, and guanine instead of cytosine. Because complements can contain nucleic acid sequences that form optimal interactions with the parent nucleic acid molecule, such complements can bind to the parent molecule with high affinity.

[0056] With respect to single-stranded nucleic acids, particularly oligonucleotides, the term “specifically hybridizes” means the relationship between two single-stranded nucleotide molecules having sufficiently complementary sequences that enable such hybridization under predetermined conditions commonly used in the art (sometimes referred to as “substantially complementary”). In particular, this term substantially excludes the hybridization of oligonucleotides with single-stranded nucleic acids with non-complementary sequences and refers to the hybridization of oligonucleotides with substantially complementary sequences contained within the single-stranded DNA or RNA molecules of the present invention. For example, specific hybridization can refer to sequences that hybridize to any lymphoid abnormality-specific marker nucleic acid but not to other nucleotides. Such markers include, for example, the lymphoid abnormality-specific markers shown in the table contained herein. Suitable conditions that enable the specific hybridization of single-stranded nucleic acid molecules with different complementarities are well known in the art.

[0057] For example, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules with specific sequence homology is shown 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)-Double structure 600 / #bp As an example of the above formula, if we use [Na+]=[0.368], 50% formamide, GC content of 42%, and average probe size of 200 bases, then T m The temperature will be 57°C. mThe temperature decreases by 1-1.5°C for every 1% decrease in homology. Therefore, targets with approximately 75% or more sequence identity will be observed using a hybridization temperature of 42°C. The stringency of hybridization and washing depends mainly on the salt concentration and temperature of the solution. Generally, to maximize the annealing rate of the probe and target, hybridization is usually performed at a calculated T of the hybrid. m The process is carried out under salt and temperature conditions 20-25°C lower. The washing conditions should be as severe as possible depending on the degree of probe identity to the target. Generally, the washing conditions are hybrid T m The temperature is selected to be approximately 12-20°C lower than the above. In a specific context, in the nucleic acids of the present invention, moderate stringency hybridization is defined as hybridization with 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg / ml denatured salmon sperm DNA at 42°C, followed by washing with 2X SSC and 0.5% SDS at 55°C for 15 minutes. High stringency hybridization is defined as hybridization with 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg / ml denatured salmon sperm DNA at 42°C, followed by washing with 1X SSC and 0.5% SDS at 65°C for 15 minutes. High stringency hybridization refers to hybridization performed at 42°C using 6X SSC, 5X Denhardt's solution, 0.5% SDS, and 100 μg / ml denatured salmon sperm DNA, followed by washing with 0.1X SSC and 0.5% SDS at 65°C for 15 minutes.

[0058] As used herein, the term “oligonucleotide” is defined as a nucleic acid molecule comprising two or more ribo or deoxyribonucleotides, preferably three or more ribo or deoxyribonucleotides. The exact size of an oligonucleotide depends on various factors and the specific application and use of the oligonucleotide. An oligonucleotide, including probes and primers, can be any length from three nucleotides to the full length of a nucleic acid molecule and explicitly includes any possible number of consecutive nucleic acids from three to the full length of a polynucleotide. Preferably, an oligonucleotide is at least about 10 nucleotides long, more preferably at least 15 nucleotides long, and more preferably at least about 20 nucleotides long.

[0059] Here, "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 can anneal to or specifically hybridize with a nucleic acid having a complementary sequence to the probe. Probes may be single-stranded or double-stranded. The exact length of a probe depends on many factors, including temperature, the source of the probe, and the application 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 specified rs number related to a single nucleotide polymorphism available in the dbSNP database) typically contains 15–25, 15–35, 20–50, or 100+ nucleotides, but may contain fewer nucleotides if the SNV site is present in the probe. The probes described herein are selected to be complementary to different strands of a particular target nucleic acid sequence. That is, the probe must have sufficient complementarity to "specifically hybridize" or anneal to each target strand under a predetermined set of conditions. Therefore, the probe sequence does not need 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, while the rest of the probe sequence is complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be scattered throughout the probe, as long as the probe sequence is sufficiently complementary to the target nucleic acid sequence and can anneal to it specifically.

[0060] A "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 a suitable environment, can function as an initiator for template-dependent nucleic acid synthesis. Given a suitable nucleic acid template, a suitable nucleic acid nucleoside triphosphate precursor, a polymerase enzyme, a suitable coenzyme, and suitable conditions such as temperature and pH, the primer can be extended at its 3' end by the addition of nucleotides through the action of polymerase or a similar enzyme, yielding 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, or longer. The primer must have sufficient complementarity to the desired template to facilitate the synthesis of the desired extension product; that is, it must be able to anneal to the desired template strand in a manner sufficient to provide the 3' hydroxyl portion of the primer to a suitable juxtaposition for use in initiating synthesis by polymerase or a similar enzyme. The primer sequence does not necessarily have to exhibit precise complementarity to the desired template. For example, a non-complementary nucleotide sequence may be fused to the 5' end of a complementary primer. Alternatively, non-complementary bases may be scattered within the oligonucleotide primer sequence. However, it is a requirement that the primer sequence has sufficient complementarity to the desired template chain sequence and functionally provides a template-primer complex for the synthesis of the extension product.

[0061] Polymerase chain reaction (PCR) is described in U.S. Patents 4,683,195, 4,800,195, and 4,965,188, the entirety of which is incorporated herein by reference.

[0062] "siRNA" refers to a molecule involved in RNA interference processes for sequence-specific post-transcriptional gene silencing or gene knockdown by providing small interfering RNA (siRNA) homologous to the sequence of a target gene. Small interfering RNA (siRNA) is synthesized in vitro or produced by cleavage from long dsRNA by ribonuclease III and mediates the degradation of sequence-specific mRNA. Preferably, the siRNA of the present invention is chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. siRNA can be synthesized as two separate complementary RNA molecules or as a single RNA molecule having two complementary regions. Commercial suppliers of synthetic RNA molecules or reagents include Applied Biosystems (Foster City, California, USA), Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colorado, USA), Pierce Chemical (part of Perbio Science, Rockford, Illinois, USA), Glen Research (Sterling, Virginia, USA), ChemGenes (Ashland, Massachusetts, USA), and Cruachem (Glasgow, UK). Specific siRNA constructs for inhibiting lymphangiomatosis mRNA may be, for example, 15-35 nucleotides in length, or more typically, about 21 nucleotides in length.

[0063] A “vector” is a single-stranded or double-stranded circular nucleic acid molecule that can infect, transfect, or transform cells and replicate independently or within the genome of a host cell. Circular double-stranded nucleic acid molecules can be cleaved by treatment with restriction enzymes, thereby linearizing them. Knowledge of vectors, restriction enzymes, and the nucleotide sequences targeted by restriction enzymes is readily available to those skilled in the art and includes any replicon such as plasmids, cosmids, bacmids, phages, or viruses, to which another genetic sequence or element (either DNA or RNA) can be attached, resulting in replication of the attached sequence or element. The nucleic acid molecules of the present invention can be inserted into a vector by cleaving the vector with a restriction enzyme and ligating the two pieces. Those skilled in the art will be well aware that when cloning gene regions containing duplications or deletions, upstream and downstream flanking sequences of the affected region of appropriate length (e.g., between 50 and 100 or more nucleotides) will be employed in the cloning process. Such vectors are useful, for example, in cell lines for studying the effects of such changes on the encoded protein.

[0064] Many techniques are available to those skilled in the art to facilitate the transformation, transfection, and 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 various techniques such as 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, and PEG fusion.

[0065] A "promoter element" is a nucleotide sequence incorporated into a vector that, upon entering a suitable cell, facilitates the binding of transcription factors and polymerases, and subsequently facilitates the transcription of a portion of the vector DNA into mRNA. In one embodiment, the promoter element of the present invention precedes the 5' end of a lymphoid system abnormality-specific marker nucleic acid molecule, and the latter is transcribed into mRNA. Subsequently, the host cell's machinery translates the mRNA into polypeptides. The promoter element can constitutively or inductively express the desired coding region.

[0066] Those skilled in the art will recognize that nucleic acid vectors may contain nucleic acid elements other than promoter elements and markers specific to lymphoid abnormalities. These other nucleic acid elements include, but are not limited to, nucleic acid sequences encoding the origin of replication, ribosome binding sites, drug resistance enzymes or amino acid metabolizing enzymes, and nucleic acid sequences encoding secretory signals, localization signals, or signals useful for polypeptide purification.

[0067] A "replicon" is any genetic element that is largely self-regulating and capable of replication, such as plasmids, cosmids, bacmids, plastids, phages, and viruses. Replicons are either RNA or DNA, and can be single-stranded or double-stranded.

[0068] An "expression operon" refers to a nucleic acid segment that contains transcriptional and translational regulatory sequences such as promoters, enhancers, translation initiation signals (such as ATG codons and AUG codons), polyadenylation signals, and terminators, and has the potential to promote the expression of polypeptide coding sequences in host cells or organisms.

[0069] In this specification, the terms “reporter,” “reporter system,” “reporter gene,” and “reporter gene product” mean an operational genetic system comprising a gene encoding a product in which, upon expression, a reporter signal is readily measurable by methods such as biological assays, immunoassays, radioimmunoassays, or colorimetric, fluorescence, or chemiluminescence methods. The nucleic acid may be RNA or DNA, linear or circular, single-stranded or double-stranded, antisense or sense polarity, and is operationally coupled to regulatory elements necessary for the expression of the reporter gene product. The necessary regulatory elements vary depending on the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, and include, but are not limited to, promoters, enhancers, translational control sequences, poly-A addition signals, and transcription termination signals.

[0070] The introduced nucleic acid may or may not be integrated (covalently bonded) to 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 plasmid. Alternatively, the introduced nucleic acid may be integrated to the nucleic acid of the recipient cell or organism, stably maintained in that cell or organism, and then inherited or passed on to the cells or organisms of the recipient cell or organism's offspring. Finally, the introduced nucleic acid may only be present transiently in the recipient cell or host organism.

[0071] A "selectable marker gene" is a gene that, when expressed, confers a selectable phenotype, such as antibiotic resistance, to transformed cells.

[0072] "Operatively linked" means that the regulatory sequences necessary for the expression of the encoded sequence are positioned within the DNA molecule at the appropriate locations relative to the encoded sequence, resulting in the expression of the encoded sequence. This same definition may also apply to the placement of transcription units and other transcriptional regulatory elements (e.g., enhancers) in expression vectors.

[0073] A "recombinant organism" or "genetically modified organism" refers to an organism that possesses a new combination of genes or nucleic acid molecules. These new combinations of genes or nucleic acid molecules can be introduced into organisms using a wide range of nucleic acid manipulation techniques available to those skilled in the art. An "organism" is any organism consisting of at least one cell. Organisms range from simple ones like a single eukaryotic cell to complex ones like mammals. Therefore, the term "recombinant organism" includes not only recombinant cells but also eukaryotes and prokaryotes. Examples of transgenic organisms include zebrafish and mice.

[0074] In this specification, the terms “isolated protein” or “isolated and purified protein” may be used. This term primarily refers to proteins produced by the expression of isolated nucleic acid molecules of the present invention. Alternatively, this term may refer to proteins that are sufficiently isolated from other naturally related proteins to exist in a “substantially pure” form. “Isolated” does not exclude the presence of artificial or synthetic mixtures with other compounds or materials, or impurities that do not interfere with the basic activity, but which may be present, for example, due to incomplete purification, addition of stabilizers, or incorporation into immunogenic or pharmaceutically acceptable formulations.

[0075] A "specific binding pair" consists of a specific binding member (SBM) and a binding partner (BP) that possess specific specificity to each other and, under normal conditions, preferentially bind to each other over other molecules. Examples of specific binding pairs include antigens and antibodies, ligands and receptors, and complementary nucleotide sequences. Those skilled in the art will know of many other examples. Furthermore, the term "specific binding pair" also applies when either or both of the specific binding member and / or binding partner constitute part of a larger molecule. In embodiments where the specific binding pair consists of nucleic acid sequences, they are of a length that allows them to hybridize with each other under assay conditions, preferably 10 nucleotides or more, more preferably 15 or 20 nucleotides or more.

[0076] A “sample,” “patient sample,” or “biological sample” generally refers to a sample that can be tested for specific molecules, preferably lymphoid abnormality-specific marker molecules such as the markers shown in the table provided below. Samples include, but are not limited to, cells, blood, serum, plasma, urine, lymph, saliva, tears, and pleural fluid.

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

[0078] In this specification, the terms “drug” and “test compound” are used interchangeably and refer to chemical compounds, mixtures of chemical compounds, biological macromolecules, or extracts made from biological materials such as cells and tissues of bacteria, plants, fungi, and animals (especially mammals). Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide / DNA complexes, and any nucleic acid-based molecules that exhibit the ability to modulate the activity of SNV-containing nucleic acids or the proteins encoding them as described herein. Exemplary drugs include, but are not limited to, at least one MEK inhibitor. Additional drugs include those listed in Table 1-2. Drugs are evaluated for their 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 for a disease in a mammal, including humans, and includes inhibiting the disease or its progression, suppressing or delaying the disease or its progression, preventing its onset, partially or completely alleviating the disease, preventing the onset of the disease, or preventing the recurrence of symptoms of the disease. An exemplary treatment includes administering at least one MEK inhibitor, or at least one of the agents listed in Tables 1-2, in an effective dose.

[0080] The terms “inhibition” or “suppression” mean a reduction or cessation of any event (such as ligand binding of a protein), or a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the occurrence, degree, or possibility of that characteristic. “Reduction” or “inhibition” means reducing, decreasing, or cessating activity, function, and / or amount compared to a baseline. The suppression or reduction does not need to be complete. For example, in one embodiment, “reduction” or “inhibition” means the ability to cause an overall reduction of 20% or more. In another embodiment, “reduction” or “suppression” means the ability to cause an overall reduction of 50% or more. In yet another embodiment, “reduction” or “suppression” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0081] An "inhibitor" is a drug that slows down or prevents a specific chemical reaction, signaling pathway, or other process, or a drug that reduces the activity of a specific reactant, catalyst, or enzyme.

[0082] The terms "patient" and "subject" are used interchangeably to refer to mammals, including humans.

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

[0084] "MEK inhibitors" or "MEK / ERK inhibitors" refer to drugs 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 also other signaling pathway processes that govern cellular homeostasis or activity.

[0085] Diagnosing patients with lymphatic system abnormalities In some embodiments, patients with lymphatic abnormalities are diagnosed based on the presence of SNVs after obtaining genotypic sequence information from a biological sample obtained from the patient. In some embodiments, patients with lymphatic abnormalities have 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 that are associated with lymphatic abnormalities. In some embodiments, this 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 identifying SNVs present in a specific subject may be generated from experimental data. In some embodiments, a report identifying recommended treatments for lymphatic abnormalities may be generated based on data of SNVs(s) identified using genotype sequence information.

[0087] 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 in linkage disequilibrium with SNVs in genes selected from PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, and CBL, guides the selection of treatment methods for the patient after obtaining genotyping sequence information from biological samples 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 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 influence or guide the patient's treatment choices after obtaining genotyping information from a biological sample obtained from the patient.

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

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

[0090] For example, nucleic acids containing lymphoid anomaly-associated SNVs can be used as probes to detect the presence and / or expression of lymphoid anomaly-specific markers. Methods by which lymphoid anomaly-associated marker nucleic acids can be used as probes for such assays include, but are not limited to, (1) in situ hybridization, (2) Southern hybridization, (3) Northern hybridization, and (4) assorted amplification reactions such as polymerase chain reaction (PCR).

[0091] Furthermore, assays for detecting SNVs associated with lymphatic system abnormalities, or the proteins encoded by them, can be performed on any type of biological sample, including body fluids (blood, urine, serum, gastric lavage fluid, etc.), all types of cells (brain cells, leukocytes, mononuclear cells, etc.), and body tissues.

[0092] Using lymphatic vessel abnormality-related SNV-containing nucleic acids, vectors expressing them, lymphatic vessel abnormality-related SNV-containing marker proteins, and anti-lymphatic vessel abnormality-specific marker antibodies, lymphatic vessel abnormality-related SNVs can be detected in body tissues, cells, or body fluids, and the expression of lymphatic vessel abnormality-related SNV-containing marker proteins can be altered to detect and diagnose lymphatic vessel abnormalities.

[0093] This includes methods for detecting and / or diagnosing lymphatic abnormalities based on lymphatic abnormality-associated SNVs. These methods may include detecting lymphatic abnormality-associated SNVs, where the SNV-containing nucleic acids in a sample are first amplified, for example, using PCR, to increase the amount of template compared to other sequences present in the sample. This allows for highly sensitive detection of the target sequence if it is present in the sample. This initial step can be avoided by using highly sensitive array techniques, which are becoming increasingly important in the art.

[0094] On the other hand, new detection techniques overcome this limitation, enabling the analysis of small sample volumes containing as little as 1 μg of total RNA. Unlike conventional fluorescence techniques, resonant light scattering (RLS) technology uses biotin-labeled hybridized targets and anti-biotin antibodies to detect small amounts of mRNA via multiple reads. Alternatively, as an alternative to PCR amplification, plane wave waveguide (PWG) technology is used to improve the signal-to-noise ratio and reduce background interference. Both technologies are commercially available from Qiagen Inc. (USA).

[0095] Thus, using any of the above techniques, the expression of SNV markers associated with lymphatic system abnormalities can be detected or quantified, and the lymphatic system abnormality or its risk of developing can be diagnosed accordingly.

[0096] Management of patients with lymphatic system abnormalities Understanding the role that the lymphoma-associated SNVs described herein play in modulating the phenotype of lymphoma will facilitate the reuse of existing therapies useful for treating lymphoma, as well as the development of new therapies. In some embodiments, the present invention includes 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 Table 1-2) to a patient with lymphoma.

[0097] In some embodiments, patients with lymphatic abnormalities requiring treatment 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 in conjunction with clinical symptoms to diagnose lymphatic abnormalities. In some embodiments, biopsies are performed to diagnose lymphatic abnormalities. In some embodiments, lymphatic abnormalities are diagnosed based on lymphatic overgrowth. In some embodiments, lymphatic abnormalities are diagnosed based on abnormal formation of lymphatic vessels. In some embodiments, lymphatic abnormalities are diagnosed based on chyle exudate, including pericardial fluid, pleural fluid, or peritoneal fluid.

[0098] In some embodiments, patients with lymphatic abnormalities to be treated are diagnosed according to the diagnostic methods described herein.

[0099] In some embodiments, one or more MEK inhibitors (e.g., one or more agents in Tables 1-2; Example II) are useful in the preparation of pharmaceuticals for treating lymphatic disorders. One or more agents may be formulated together with pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials are preferably non-toxic and do not inhibit the efficacy of the active ingredient. The exact properties of carriers and 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 disorders may be used to select specific agents for human treatment.

[0100] The drugs listed in Tables 1 and 2 are useful for treatment, but are not limited to these. Some drugs are listed in both Tables 1 and 2, and there is no significance to a drug not being listed in both tables. [Table 1] TIFF0007901128000002.tif152141TIFF0007901128000003.tif171141TIFF0007901128000004.tif122141

[0101] Table 2 provides drugs that can be used alone or in combination with any of the drugs in Table 1 or Table 2 to treat lymphatic abnormalities. [Table 2] TIFF0007901128000006.tif106141 [ka]

[0102] To treat individuals with lymphatic vascular abnormalities, or to alleviate the signs and symptoms of the disease, a combination of appropriate gene-targeting agents disclosed herein can provide therapeutic benefits to patients. Such agents should be administered in effective doses.

[0103] Once a genetic mutation is identified, therapeutic approaches are devised to modulate the biological and signaling pathways affected by that gene. For example, if inhibiting the MAPK (MEK1 / MEK2) and ERK pathways is desirable, MEK inhibitors can be used alone or in combination with other MEK / ERK inhibitors. In certain embodiments, treatment involves the administration of drugs listed in Table 1 or 2, such as mTOR inhibitors, together with a PIK3K inhibitor. In other embodiments, a combination of mTOR inhibitors and MEK / ERK inhibitors provides therapeutic benefits to the patient. Another approach involves combining a PIK3K inhibitor and a MEK / ERK inhibitor to improve the symptoms of the disease. For the specific ARAF gain-of-function mutations described herein, an effective therapeutic approach consists of the administration of MEK / ERK inhibitors. The combinatorial therapies described above can act additively. In other embodiments, the combined drugs act synergistically to alleviate symptoms.

[0104] First, biological samples and genotypic information are obtained from the patient. Then, based on the genetic information obtained from the nucleic acids contained in the sample, the presence of SNVs associated with lymphatic abnormalities is evaluated, 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 clinicians with guidance on which therapeutic agents are appropriate. The total therapeutic dose or dosage (if two or more targets are modified) can be administered to the subject as a single dose or using a fractionation therapy protocol. In this protocol, multiple / separate doses are administered over a longer period, for example, administered over a period of one day to administer the daily dose, or administered over a longer period to administer over the desired period. Those skilled in the art will know that the amount of lymphoid agent required to obtain an effective dose in a subject depends on many factors, including the subject's age, weight, general health status, as well as the route of administration and the number of treatments administered. Taking these factors into consideration, those skilled in the art will adjust a particular dose to obtain an effective dose for treating an individual with lymphoid abnormalities.

[0105] The effective dose of a 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 typically range from approximately 0.001 mg to approximately 1000 mg.

[0106] In particular, in individuals suffering from more severe forms of lymphatic abnormalities, the administration of lymphatic abnormality-relieving agents is especially useful, for example, when administered in combination with conventional drugs for treating such diseases. Those skilled in the art will administer lymphatic abnormality-relieving agents alone or in combination and monitor the effects 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 agents for treating lymphatic abnormalities include systemic chemotherapy, interferon-alpha therapy, radiotherapy, or surgery to alleviate the underlying symptoms of the disease.

[0107] The administration of a pharmaceutical formulation is preferably an "effective dose," which is a sufficient amount to demonstrate a benefit to the individual. This dose prevents, alleviates, reduces, or otherwise reduces the severity of the symptoms of the lymphatic abnormality in the patient. Treatment of patients with lymphatic abnormalities with an effective dose of MEK inhibitors and / or the drugs listed in Table 1-2) may result in improved lymphatic structure, reduced pleural effusion, improved respiratory function, gradual reduction in the use of concomitant medications, or improved survival rates.

[0108] This product is formulated in the form of dosage units to facilitate administration and ensure uniformity of dosage. A dosage unit, as defined here, is a physically separated unit of the pharmaceutical preparation appropriate for the patient receiving treatment. Each dosage should contain an amount of the active ingredient calculated to produce the desired effect in combination with a selected pharmaceutical carrier. The procedure for determining appropriate dosage units is well known to those skilled in the art.

[0109] The dosage unit can be increased or decreased proportionally according to the patient's weight. The appropriate concentration for alleviating a specific medical condition can be determined by dose-to-concentration curve calculation, as is known in the art.

[0110] Pharmaceutical compositions useful for the methods of the present invention can be administered systemically in parenteral, oral solid and liquid formulations, subcutaneously, intradermally, intramuscularly, sublingually, topically, via the auricle (OTIC), buccal, conjunctiva, skin, teeth, electroosmosis, via the cervix, sinus or trachea, enterally, epidurally, infiltrate, interstitially, intraperitoneally, intra-arterially, intra-articularly, intra-biliaryly, intra-tracheally, intracardiacly, intracardiacly, intra-tracheally, intra-sternally, intrathoracically, intracerebrally, intradermally, intra-lymphaticly, intracardiacly, intraperitoneally, intra-nasally, percutaneously, respiratoryly, ophthalmally, suppositories, aerosols, topically, and via other known routes of administration. In addition to agents useful for the treatment of lymphatic disorders, pharmaceutical compositions may also contain pharmaceutically acceptable carriers and other components known to enhance and promote the administration of the agent. Thus, such compositions may optionally contain other components, such as adjuvants, such as aqueous suspensions of aluminum hydroxide and magnesium hydroxide, and / or other pharmaceutically acceptable carriers, such as saline. Other possible formulations, such as nanoparticles, liposomes, reencapsulated erythrocytes, and immunologically based systems, may also be used to deliver / administer appropriate drugs to patients according to the method of the present invention. The use of nanoparticles for delivering such drugs and available cell membrane-permeable peptide carriers is described in Crombez et al., Biochemical Society Transactions v35:p44 (2007).

[0111] The administration of drugs useful for treating lymphatic system abnormalities can be performed after successfully detecting or quantifying the expression of SNV markers associated with lymphatic system abnormalities, thereby diagnosing the abnormality or the risk of its development. Detecting or quantifying the expression of SNV markers associated with lymphatic system abnormalities allows for the selection of specific drugs to be used for treatment. Detecting or quantifying the expression of SNV markers associated with lymphatic system abnormalities can also indicate that a particular treatment is inappropriate for the subject.

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

[0113] In other embodiments, treatment for lymphatic abnormalities may be initiated based on a clinical diagnosis of the disease, and treatment may be initiated when the expression of SNV markers associated with lymphatic abnormalities is no different from that of the control.

[0114] In some embodiments, treatment is performed on patients who do not have SNVs of PTPN11, KRAS, BRAF, SOS1, ITGA9, RASA1, RAF1, RIT1, PEIZO1, EPHB4, NF1, ARAF, or CBL.

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

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

[0117] In some embodiments, the drugs administered in the treatment method include rapamycin (sirolimus), everolimus (RAD001), AZD8055, temsirolimus (CCI-779, NSC 683864), KU-0063794, MHY1485, BEZ235 (NVP-BEZ235, dactricib), PI-103, tolkinib (PP242), tacrolimus (FK506), ridafololimus (defololimus, MK-8669), and INK. 128 (MLN0128), Voxatalisib (SAR245409, XL765), Torin1, Omipaliciib (GSK2126458, GSK458), OSI-027, PF-04691502, Apitricib (GDC-0980, RG7422), GSK1059615, Jedatricib (PF-05212384, PKI-587), WYE-354, AZD2 The following are selected: 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), Palomide 529 (P529), Chrysophanic acid, etc.

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

[0119] Furthermore, the above-mentioned combinations of drugs can 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) ridafololimus and trametinib, 2) ridafololimus and selumetinib or cobimetinib, 3) BEZ235 and selumetinib, 4) omiparisib and selumetinib or trametinib, 5) everolimus and trametinib or selumetinib, 6) sirolimus, ridafololimus and selumetinib, 7) sirolimus, ridafololimus and trametinib, 8) tolkinib and trametinib; 9) BEZ235, tolkinib and trametinib, 10) sirolimus, gedatricib and trametinib.

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

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

[0122] The chromosomal regions described here include protein-coding regions, which can be used to rationally design therapeutics that modulate protein activity. Small peptide molecules corresponding to these regions can be advantageously used when designing therapeutics that effectively modulate the activity of the encoded proteins.

[0123] Molecular modeling should allow for the identification of specific organic molecules capable of binding to the active site of proteins encoded by nucleic acids, including SNVs, based on the conformational and functionally necessary key amino acid residues. Combinatorial chemistry techniques can be used to identify the molecules with the highest activity, develop these molecules iteratively, and conduct further screening cycles. In one embodiment, candidate drugs can be screened from a large library of synthetic or natural compounds. One example is an FDA-approved library of compounds for human use.Furthermore, the compound libraries include Maybridge Chemical Co. (Trevilet, Cornwall, UK), Comgenex (Princeton, New Jersey), Microsource (New Milford, Connecticut), Aldrich (Milwaukee, Wyoming), AKos Consulting and Solutions GmbH (Basel, Switzerland), Ambinter (Paris, France), Asinex (Moscow, Russia), Aurora (Graz, Astoria), BioFocus DPI, Switzerland, Bionet (Camelford, UK), ChemBridge (San Diego, California), ChemDiv (San Diego, California), Chemical Block Lt (Moscow, Russia), ChemStar (Moscow, Russia), and Exclusive Chemistry, Ltd. (Russia, Obninsk), Enamine (Ukraine, Kyiv), Evotec (Germany, Hamburg), Indofine (New Jersey, Hillsboro), Interbioscreen (Russia, Moscow), Interchim (France, Montulcon), Life Chemicals, Inc. (Connecticut, Orange), Microchemistry Ltd. (Russia, Moscow), Otava (Ontario, Toronto), PharmEx Ltd. (Russia, Moscow), Princeton Biomolecular (New Jersey, Monmouth Junction), Scientific Exchange (New Hampshire, Centre Ossie), Specs (Delft, Netherlands), TimTec (Delaware, Newark), Toronto Research Corp. (Ontario, North York), UkrOrgSynthesis (Ukraine, Kyiv), Vitas-M (Russia, Moscow), Zelinsky Institute (Russia, Moscow), and It is marketed by many 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 products such as animals, bacteria, fungi, plants (including leaves and bark), and marine samples as candidates for indicating the presence of potentially useful pharmaceuticals. It will also be understood that the pharmaceuticals being screened may be derived or synthesized from chemical compositions or artificial compounds. Several commercially available libraries can be used for screening.

[0125] Polypeptides and fragments used in drug screening may be free in solution, attached to a solid support, or present within cells. One method of drug screening utilizes eukaryotic or prokaryotic host cells stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably performing competitive binding assays. Such cells can be used in standard binding assays, either in living or fixed form. For example, the formation of a complex between the polypeptide or fragment and the drug under test can be determined, or the extent to which the formation of a complex between the polypeptide or fragment and a known substrate is inhibited by the drug under test can be investigated.

[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) that have non-functional or altered lymphoid abnormality-related genes. In some cases, transgenic organisms have 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 c.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 one or more mutations as shown in Table 3. These host cell lines, cells, or transgenic animals are deficient at the polypeptide level. The host cell lines or cells are cultured in the presence of the drug compound. For example, in a zebrafish model, the salvage of tail and / or D / V vascular structures can be evaluated. Furthermore, the induction of phosphorylation by mTOR in host cell lines may also be evaluated.

[0127] An exemplary method for drug screening is a method for identifying drugs that alter cellular signaling, such as the drugs listed in Table 1-2. This method would include the steps of: providing cells expressing at least one nucleic acid containing at least one lymphoid disorder-associated SNV; providing cells expressing a congeneral wild-type sequence corresponding to the lymphoid disorder-associated SNV; contacting the cells expressing at least one lymphoid disorder-associated SNV and the cells expressing the congeneral wild-type sequence with a test agent; and analyzing whether the test agent alters cellular signaling.

[0128] The host cells expressing lymphoid anomaly-related SNVs or their functional fragments according to the present invention provide a system for screening potential compounds or drugs for their ability to modulate the development of lymphoid anomalies. Accordingly, in one embodiment, the nucleic acid molecules of the present invention can be used to create recombinant cell lines for use in assays to identify drugs that modulate aspects of abnormal MAPK signaling associated with lymphoid anomalies and abnormal angiogenesis. Furthermore, the specification provides a method for screening compounds that can modulate the function of proteins encoded by SNV-containing nucleic acids.

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

[0130] In another embodiment, the availability of lymphoid disorder-associated modified nucleic acids makes it possible to produce strains of experimental mice possessing the modified nucleic acids of the present invention. These lymphoid disorder-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, and c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene, as well as any of the other mutations shown in Table 3. Transgenic mice expressing the lymphoid disorder-associated mutations of the present invention provide a model system for investigating the role of proteins encoded by the mutated nucleic acids in the development and progression of lymphoid disorders. Methods for introducing transgenes into laboratory mice are known to those skilled in the art. Three common methods are: 1. integration of a retroviral vector encoding the target foreign gene into an early embryo; 2. injection of DNA into the pronucleus of a newly fertilized egg; and 3. integration of genetically engineered embryonic stem cells into an early embryo. By creating the transgenic mice described above, it becomes possible to elucidate at the molecular level the role of target proteins in various processes related to the phenotype of lymphatic abnormalities. Such mice provide an in vivo screening tool for studying putative therapeutics in all animal models and are included in the present invention.

[0131] In this specification, the term “animal” is used to mean all vertebrates except humans. It also includes individual animals at all developmental stages, including embryos and fetuses. A “transgenic animal” is an animal that contains one or more cells that have been directly or indirectly altered or received genetic information through intentional genetic manipulation at the subcellular level, such as targeted recombination, microinjection, or infection with recombinant viruses. 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 recombinant DNA molecules. These molecules may be targeted at specific loci, randomly incorporated into chromosomes, or replicated outside of chromosomes. A “germline transgenic animal” is a transgenic animal in which genetic modification or genetic information has been introduced into germline cells, thereby conferring the ability to transmit genetic information to offspring. If the offspring actually possess some or all of the alteration or genetic information, then those offspring are also transgenic animals.

[0132] Modifications to genetic information can be heterogeneous to the recipient's animal species, heterogeneous only to a specific individual recipient, or they may be genetic information already present in the recipient. In the last case, the modified or introduced gene may be expressed differently from the native gene. Such modified or foreign genetic information would include the introduction of modified lymphatic abnormality-related nucleotide sequences.

[0133] DNA used to modify target genes can be obtained by a wide range of techniques, including but not limited to isolation from genomic sources, preparation of cDNA from isolated mRNA templates, direct synthesis, or combinations thereof.

[0134] One of the cells to be targeted for introduction is embryonic stem cells (ES). ES cells can be obtained from pre-implantation embryos cultured in vitro (described in 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).) The transgene can be efficiently introduced into ES cells by standard techniques such as DNA transfection or introduction via retroviruses. As a result, the transformed ES cells can then be combined with blastocysts of non-human animals. The introduced ES cells then colonize the embryo and contribute to the germ line of the resulting chimeric animals.

[0135] One approach to the problem of determining the contribution of individual genes and their expression products is to use lymphoid disorder-related genes containing isolated mutations 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 that uses target homologous recombination to insert specific changes into alleles of chromosomes to inactivate any gene region or change it to a desired mutation. However, plasmid-chromosome homologous recombination is reported to be detected only at a frequency of 10 -6 ~10 -3 compared to extrachromosomal homologous recombination that occurs at a frequency close to 100%. Non-homologous plasmid-chromosome interactions occur at a frequency 10 5 times to 10 2 times higher than equivalent homologous insertions.

[0137] To overcome the low rate of targeted recombination in mouse ES cells, various strategies have been developed to detect or select rare homologous recombinants. One approach to detecting homologous change events involves screening a pool of transformed cells using polymerase chain reaction (PCR) to detect homologous insertions, followed by screening individual clones. Positive genetic selection methods have also been developed, which involve constructing marker genes that are activated only when homologous insertions occur, allowing for the direct selection of these recombinants. One of the most powerful approaches developed for selecting homologous recombinants is positive-negative selection (PNS), developed for genes for which direct selection of modifications does not exist. PNS 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 effective herpes drugs such as ganciclovir (GANC) or (1-(2-deoxy-2-fluoro-BD-arabinofuranosyl)-5-ioduracil (FIAU) with the herpes simplex virus thymidine kinase (HSV-TK) gene. This counter-selection can increase the number of homologous recombinants in the surviving transformants. By utilizing mutated lymphoid abnormality-associated nucleic acids as targeted insertion cassettes, a means is provided to detect successful insertions, for example, by the acquisition of immunoreactivity against immunologically specific antibodies to the polypeptide encoded by the EPHB4 nucleic acid, thus facilitating the screening / selection of ES cells with the desired genotype.

[0138] In this specification, a knock-in animal is, for example, an animal in which an endogenous mouse gene has been replaced with a human lymphatic abnormality-related gene of the present invention. Such a knock-in animal provides an ideal model system for studying the development of lymphatic system abnormalities.

[0139] This specification describes how the expression of mutated lymphoid disorder-associated nucleic acids, fragments thereof, or lymphoid disorder-associated fusion proteins can be targeted in a "tissue-specific" or "cell-type-specific" manner using vectors in which nucleic acid sequences encoding all or part of a lymphoid disorder-associated nucleic acid are operably ligated to regulatory sequences (e.g., promoters and / or enhancers) that direct the expression of the encoded protein in a specific 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 are described herein. Alternatively, the transgene may be under the control of an inducible promoter that functions tissue-specifically or "systemically".

[0140] The nucleic acid sequences encoding lymphoid abnormality-associated mutants of the present invention may be operablely ligated to various different promoter sequences for expression in transgenic animals. Such promoters include, but are not limited to, prion gene promoters such as the Thy-1 promoter in hamsters and mice; PGK promoters; or CMV promoters for expressing transgenes in desired cell types.

[0141] Methods of using the transgenic mice of the present invention are also provided herein. Transgenic mice into which nucleic acids, including mutated lymphoid disorder-associated nucleic acids or proteins encoding them, have been introduced are useful, for example, in developing methods for screening therapeutic agents to identify those that can modulate the development of lymphoid disorders.

[0142] Detection products and kits This includes compositions or products useful for detecting lymphoid SNVs. For example, lymphoid SNV-containing nucleic acids, vectors expressing them, lymphoid SNV-containing marker proteins, and anti-lymphoid aberration-specific marker antibodies are products that can detect complex mutations such as 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, and c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene. The scope includes nucleic acid probes of sufficient length and characteristics to detect SNVs such as 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, and c.1236+4A>G and c.289T>G:p.C97G in the ITGA9 gene. The detection products may be labeled for detection.

[0143] Any product useful for detecting lymphoid system abnormalities-related SNVs can be incorporated into the kit. Any product useful for treating lymphoid system abnormalities can be incorporated into the kit. Kits containing such detection and therapeutic products are included. The kit may include one or more lymphoid system abnormality-related SNV-specific marker polynucleotides or one or more aggregates of such markers immobilized on a solid support, gene chip, oligonucleotides, polypeptides, peptides, antibodies, labels, markers, reporters, pharmaceutically acceptable carriers, physiologically acceptable carriers, instructions for use, containers, dosing containers, assay substrates, or any combination thereof.

[0144] The following examples are provided to illustrate specific embodiments of the present invention. They 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 lymphangiogenesis defects. To identify the genetic basis of CCLA, whole exome sequencing was performed on DNA samples from seven patients. All of these patients had atypical lymphatic vessels imaged by dynamic contrast magnetic resonance lymphangiography (DCMRL), a new advanced imaging technique that allows for better diagnosis of this group. In five patients, missense, nonsense, splice changes, and coding indel mutations that could explain the phenotype were examined. The findings were filtered to exclude synonymous mutations, mutations with minor allele frequencies (MAF) greater than 0.5%, and mutations already identified in the control group in the in-house exome mutation database. Relevant candidates were subjected to manual curation. As a result, one somatic mutation and five de novo missense mutations were identified in four genes involved in the Ras / MAPK signaling pathway (PTPN11, KRAS, BRAF, SOS1) (Table 3). Furthermore, in one patient with primary lymphedema and retrograde lymphatic flow, two heterozygous mutations were discovered in ITGA9: c.1236+4A>G and c.289T>G:p.C97G. ITGA9 encodes intergrin α9, a cell surface glycoprotein that mediates cell-to-cell adhesion and cell-to-matrix interactions. Integrin α9 binds to VEGF-C, and inactivation of ITGA9 leads to thoracic wall formation and inhibits lymphatic valve formation in mice. The sequences of these proteins are listed in the NCBI database and are as follows: a) PTPN11's c.1504T>G:pS502A, NCBI reference sequence located at S502::NP_002825.3 b) PTPN11 c.1510A>G:pM504V NCBI reference sequence present in M504: NP_002825.3 c) PTPN1 c.1507G>C:pG503R NCBI reference sequence present in G503: NP_002825.3 d) KRAS c.35G>A:pG12D NCBI reference sequence located in G12: NP_203524.1 e) BRAF's c.1403T>C:pF468S NCBI reference sequence located at F468: NP_004324.2 f) SOS1's c.2536G>A:pE846K NCBI reference sequence located at 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 the exon-intron junction, and also found within introns. [Table 3] TIFF0007901128000009.tif49141TIFF0007901128000010.tif37141TIFF0007901128000011.tif206141TIFF0007901128000012.tif196141TIFF0007901128000013.tif206141TIFF0007901128000014.tif199141TIFF0007901128000015.tif194141TIFF0007901128000016.tif197141TIFF0007901128000017.tif210140TIFF0007901128000018.tif197141TIFF0007901128000019.tif210139TIFF0007901128000020.tif199141TIFF0007901128000021.tif210141TIFF0007901128000022.tif210140TIFF0007901128000023.tif197141TIFF0007901128000024.tif196141TIFF0007901128000025.tif210139TIFF0007901128000026.tif205141TIFF0007901128000027.tif64141

[0147] RASopathies are a group of genetically heterogeneous disorders involving mutations in the Ras / MAPK pathway, including PTPN11, SOS1, RAF1, KRAS, HRAS, MAP2K1, MAP2K2, NRAS, CBL, SHOC2, BRAF, RIT1, A2ML1, SPRED1, and NF1. These disorders include Noonan syndrome (NS), Cardiofaciocutaneous syndrome (CFC), neurofibromatosis type 1 (NF1), Noonan syndrome with lentigo multifocals (NSML), Costello syndrome (CS), Legius syndrome, and Regius syndrome. Lymphatic deficiencies in NS have been reported in a subset of NS patients, but their severity, location, and onset vary. A literature review identified 52 prenatal and postnatal patients presenting with the clinical features and lymphatic deficiencies of Noonan syndrome or Noonan-like syndrome, including pleural effusion, pericardial effusion, pyorrhea, edema, lymphangiectasia, and lymphedema. We identified the PTPN11 mutations that explain their disease. Therefore, therapeutic interventions targeting the ras / MAPK ERK pathway should reverse the disease phenotype in these patients, similar to what we have shown with ARAF gene mutations. Thus, our finding that patients with central lymphatic disorders 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] In the cell line Ea.hy926, a cell model developed to investigate the effects of mutations that may activate ERK, wild-type and mutant proteins were expressed using retroviruses. The Ea.hy926 cell line is a fusion of human umbilical vein endothelial cells and a melanoma cell line. The Ea.hy926 cell line is immortalized, retaining some endothelial cell characteristics. Using this cell line, we successfully demonstrated the effects of ARAF gene mutations, which cause central lymphatic abnormalities, on cells. Furthermore, we succeeded in reversing 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 wild-type (WT) or mutant BRAF or PTPN11 protein. Normalized by protein quantity, this experiment shows that cells expressing mutant BRAF contain more than five times the amount of phosphorylated ERK compared to cells expressing wild-type BRAF. Mutant BRAF is expressed more highly than WT, as previously observed with ARAF.

[0150] Figure 2 shows changes in cell morphology in cells expressing the F486S BRAF mutant. Filamentous actin, revealed by phalloidin staining, is present throughout the cell body of cells expressing wild-type BRAF. However, in cells expressing the BRAF mutant, actin was mostly confined to the periphery. This behavior is consistent with previous observations regarding cells expressing the ARAF mutant. VE-cadherin staining is mostly intracellular in cells expressing either wild-type (WT) or mutant BRAF. Previous results using ARAF suggest that ERK activation is induced in the Ea.hy926 cell line even with WT BRAF expression. As a result, although WT BRAF does not change the overall cell morphology, VE-cadherin is internalized. Figure 3 highlights the effect of ERK activation on both VE-cadherin localization and cell morphology. When cells expressing WT or mutant BRAF are treated, cell surface staining of VE-cadherin dramatically increases, and filamentous actin in the cell body increases. In this experiment, the morphological differences between cells expressing WT and the mutant were not very pronounced, which may be due to variability between experiments or because the culture time and cell density were increased in this experiment compared to Figure 2. No changes in cell morphology or VE-cadherin distribution were observed in cells expressing the PTPN11 mutant.

[0151] To find more effective inhibitors, cells expressing WT and mutant BRAF or PTPN11 were treated with various MEK inhibitors. See Figures 4 and 5. We performed titration curves with PD0352971, pimacertib, and rifametinib, using trametinib as a control. As expected, each inhibitor was able to reduce intracellular pERK levels in a dose-dependent manner. Notably, in this experiment, PTPN11 mutants did not appear to activate ERK beyond the levels seen in WT PTPN11. BRAF F486S consistently induced ERK activation above WT levels, although the specific multiplier of increase varied from gel to gel.

[0152] Zebrafish models of lymphatic vessel diseases Clones were incorporated into a vector containing a Tol2 transposase flanking site using a gateway system (Invitrogen, Kwan 2007, Villefranc 2007 17937395, 17948311) to enable integration into the genome (Kawakami 1999, 10564832). Approximately 10 pl of 25 ng / ul of Tol2 mRNA and vector DNA was injected into the first cells of fresh fertilized eggs. The construct was injected into Casper fish expressing mrc1a:GFP, and lymphatic vessels were visualized. Confocal scans were performed using a Zeiss LSM710 confocal microscope and Zen software. Confocal Z-stack images were superimposed using the maximum intensity projection function of Zeiss Zen software. Images were edited using imageJ (Fiji) {Schindelin 2012} and PowerPoint (Microsoft).

[0153] To evaluate whether mutant human KRAS G12D affects lymphangiogenesis in zebrafish, both mutant and wild-type genes were cloned after a lymphatic vessel-specific mrc1a promoter (Jung 2017, PMID:28506987) and ligated to the mCherry fluorescent via the v2a autocatalytic cleavage site (Provost 2007, 17941043). A flanking Tol2 transposase site was used for genome integration. When tol2 mRNA was injected into this construct, phenotypic analysis at 7 dpf (a few days post-fertilization) revealed small patches of lymphatic cells expressing the transgene and the mCherry marker in most injected fish.

[0154] We examined the phenotype of the trunk thoracic duct, one of the largest vessels at this stage, where the expression of the transgenic clone was most consistent. While WT expression of KRAS did not affect lymphatic vessel development, KRAS G12D caused dilation and expansion of the thoracic duct and appeared to fuse with the ventral cardinal vein.

[0155] In additional studies, we further identified genes / mutations in the RAS / MAPK pathway that explain lymphatic abnormalities and offer 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 pathogenesis of these disease groups and the importance of RAS / MAPK pathway mutations in lymphatic disorders. Furthermore, one homozygous missense mutation was found in patients with lymphedema and lymphatic conduction disorders. In this study, we present data showing that therapeutic intervention of the RAS / MAPK pathway can reverse the increased ERK1 / 2 activity and lymphatic phenotype induced by this mutation in zebrafish, similar to what we have previously shown with ARAF mutations.

[0156] To better understand and study the effects of potential ERK-activating mutations, we developed a cell model using primary lymphatic endothelial cells instead of established cell lines, capturing the characteristics of mutations in disease-associated cell types. We expressed wild-type and mutant proteins in human dermal lymphatic endothelial cells (HDLECs) using retroviruses. 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, a finding recently published in Nature Medicine. Furthermore, testing seven additional MEK / ERK inhibitors in this model revealed biochemical and morphological reversals of the ARAF mutation-induced effects, comparable to those of trametinib, for PD0325901 (Figure 6), CI1040 / PD184352 (Figure 7), pimacerutib (Figure 8), TAK-733 (Figure 9), AZD8330 (Figure 10), rifametinib (Figure 11), and urixertinib (Figure 12).

[0157] Figures 6-11 show the effects of various drugs (shown in the figures) on HDLECs expressing the ARAF mutation S214P. All drugs tested were sufficient to reverse the morphological differences induced by ARAF-S214P, indicating greater accumulation of VE-cadherin in the cell membrane. Green indicates HA staining for ARAF-expressing cells, and red indicates VE-cadherin staining.

[0158] Ulixertinib is an orally effective ERK1 / 2 inhibitor. Previous studies have shown that p-ERK1 / 2 levels increase in various cancer cell lines after urixertinib administration. However, phosphorylation of RSK (a substrate of the ERK1 / 2 protein) decreases, which is consistent with sustained ERK1 / 2 inhibition. As shown in the upper panel of Figure 12, urixertinib treatment suppresses the phosphorylation of RSK3, an ERK substrate. Furthermore, as shown in the lower panel, urixertinib can almost completely restore and rescue VE-cadherin loss from the intercellular junctions of HDLECs expressing ARAF-S214P at a concentration of 300 nM (Figure 12; green indicates ARAF-expressing cells with HA staining, red indicates VE-cadherin staining, and white indicates nuclei with DAPI staining). Similarly, when BRAF-F486S is expressed in HDLEC cells, significant accumulation of VE-cadherin between adjacent cells is eliminated (Figures 13-14), resulting in a phenotype essentially similar to that of ARAF mutations. As expected, treatment with urixertinib (Figure 13) or trametinib (Figure 14) can rescue the phenotype, with trametinib (upper panel of Figure 14) suppressing the increase in ERK phosphorylation. Furthermore, HDLEC cells expressing RAF1 or KRAS mutations showed similar morphological changes and similarly elevated p-ERK levels, which could be normalized by trametinib and urixertinib, respectively (Figures 15-16). On the other hand, RIT1 mutations induce weak activation of p-ERK (Figure 17).

[0159] Insights into the effects of mutations on lymphangiogenesis were obtained using a spheroid sprouting assay (3D lymphangiogenesis assay) with HDLECs. HDLECs expressing EPHB4-ins, EPHB4-R763Q, or EPHB4-K885 showed enhanced lymphangiogenic capacity compared to HDLECs expressing EPHB4-WT, as measured by sprout length in a 3D lymphospheroid sprouting assay performed in the presence of vascular endothelial growth factor C (VEGFC), with or without ephrin B2 (Figure 18, top and bottom left panels). Both rapamycin and OSI-027, potent and selective inhibitors of mTORC1 and mTORC2, can save the increased sprouting of mutants (Figure 18, bottom right panel).

[0160] In a zebrafish model, overexpression of the KRAS-G12D mutant in lymphatic vessels, rather than wild-type KRAS, resulted in thoracic duct dilation and significant edema. Furthermore, we 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, pimacertib, TAK-733, AZD8330, and PD0325901 reduced edema and improved disordered lymphatic branching (Figure 19). Additionally, treatment with NVP-BEZ235, a dual inhibitor of PI3K and mTOR, significantly reduced edematous larvae (Figure 19).

[0161] Furthermore, mutations in PTPN11 were overexpressed in a zebrafish model. Figure 20 shows that when S502A or G503R mutations of PTPN11 were expressed in a mosaic pattern, a phenotype of mild lymphatic abnormalities was observed. This included lymphatic tissue dilation and misdirection, as well as fusion of the posterior cardiac vein and thoracic duct.

[0162] Zebrafish have two RASA1 homologs, rasa1a and rasa1b. We designed gRNAs targeting both 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 Lymphatic and other vascular malformative / overgrowth disorders caused by 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,Brioar,Brorrow PEVR,Cozz, VR 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,Dellis,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 16. Joyce S, Gordon K, Brice G, Ostergaard P, Nagaraja R, Short J, Moore S, Mortimer P, Mansour S. Eur J Hum Genet 24:690–696 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. 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[0164] Example 2 Recurrent ARAF mutations cause central conduction lymphomas that can be treated with MEK inhibitors. Recent studies have shown the usefulness of sirolimus in the treatment of systemic lymphangiopathy (GLA) and central lymphangiopathy (CCLA)3-5, but the rarity of these diseases and overlapping diagnostic criteria mean that there is no clear clinical distinction between them, which hinders the development of innovative therapies6-9. GLA is defined as a multifocal lymphatic disorder involving malformations of microcystic lymphatic vessels, often presenting in multiple regions and accompanied by bone destruction9-11. CCLA, on the other hand, is a dysfunction of the thoracic duct (TD) or thoracoscopy, causing lymphatic reflux or abnormal lymphatic drainage1,12,13. Both diseases present with thoracic effusion, pleural effusion, biliary ascites, and lymphedema. The overlap of these seemingly distinct diseases suggests that a common pathway, rather than a common gene, is the cause. This means that it can lead to various clinical syndromes, suggesting that the differentiation between the diseases may be artificial. Here, we report the identification of recurrent missense mutations in ARAF underlying a severe, progressive lymphoid disease characterized by complex lymphatic abnormalities in two unrelated patients, using whole-exome sequencing (WES). This result provides a prime example of how genetic classification can categorize complex medical diseases. As a result, evidence-based treatment became possible, and in our case, a life was saved.

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

[0166] patient After obtaining approval and written informed consent from the Institutional Review Board of Children's Hospital of Philadelphia (CHOP), blood samples were obtained from the attending physician, Proband (P1), and his parents, and sequenced. Proband had severe lymphatic fluid accumulation in his chest, pericardium, abdomen, lower extremities, and genitals, and was followed up and treated at the CHOP Center for Lymphatic Imaging and Interventions. A second, unrelated adult patient (P2) was recruited through the Lymphangiomatosis & Gorham's Disease Alliance (LGDA) Patient Registry and participated along with any available family members. P1's birth and family history were normal except for a capillary malformation in the left flank, and his growth and development in early childhood were normal. At age 10, he developed swelling in his lower abdomen, thighs, scrotum, and penis. Two months later, he visited a local hospital complaining of shortness of breath and exercise intolerance. A chest X-ray revealed cardiac hypertrophy, and an echocardiogram confirmed a large pericardial effusion. Pericardiocentesis was performed, and 1 liter of cerebrospinal fluid was drained. Complete parenteral nutrition was implemented, but pericardial effusion continued, so the patient was transferred to CHOP for further management. At CHOP, dynamic contrast-enhanced magnetic resonance lymphangiography was performed as an initial assessment, confirming a large pericardial effusion and reflux from a dilated lumbar and retroperitoneal network into a dilated and winding TD towards the left denominator vein (Figure 1a, c, d). Figure 1b shows an image of an asymptomatic person for reference. Furthermore, retrograde lymphatic flow to the liver, mesentery, penis, and scrotum was observed, and retrograde lymphatic flow from the distal TD to the mediastinum and pericardium was observed (Figure 1c-e). Therefore, a stent was placed in the distal TD, and lipiodol embolization was performed to prevent abnormal lymphatic fluid accumulation in the mediastinum and pericardium. The pericardial effusion was stable, and the patient was discharged after one month. However, shortly thereafter, a large amount of fluid accumulated again, leading to respiratory distress and requiring oxygen supplementation (up to 5 liters supplied via nasal cannula). Sirolimus administration was started, and based on the trough level at which the patient could tolerate well, a dose of 2.5 mg per day was administered. Between May and November 2016, the median trough level was 11.8 (range 6.8~16.2 μg dl-1).Over a period of 1.5 years, the patient underwent multiple percutaneous interventions and surgical lymphatic procedures, including repeated thoracentesis and pleural drainage, multiple percutaneous lymphoembolizations, two bilateral surgical pleuracentesis, surgical lymphatic anastomosis of the thigh, abdomen, and retroperitoneum, and surgical ligation and embolization of the inguinal lymphatic vessels due to worsening penile and scrotal edema. Despite repeated attempts to control pericardial effusion, lymphedema of the penis, scrotum, lower extremities, and lower abdomen worsened, and the patient's condition steadily deteriorated, leading to consideration of palliative care. The final treatment was performed, and sirolimus was discontinued five months before trametinib. [Table 4]

[0167] Patient P2 was an unrelated adult woman diagnosed with lymphangiomatosis at age 31. Prior to diagnosis, P2 had experienced symptoms for many years, with significant pulmonary infiltration and multiple pleuropunctures before pleurodesis. Extensive gastrointestinal lesions were also present, requiring a specialized low-fat diet, supplementation with medium-chain triglyceride oil, and intermittent parenteral nutrition. Because of persistent symptoms of unknown cause, computed tomography and magnetic resonance imaging were performed, consistent with lymphangiomatosis occurring in the kidneys, liver, spleen, and lungs.

[0168] The patient was diagnosed with lymphangiomatosis via liver biopsy. Further treatment involved albuterol and diuretics, and the patient used an electric scooter due to fatigue and shortness of breath. No bone infiltration was observed. This patient was recruited by the Lymphangiomatosis & Gorham's Disease Alliance and, being from a non-local area, could not be followed up. Furthermore, the patient died from complications related to the underlying lymphatic disorder and was unable to participate in trials of other treatments.

[0169] WES and Bioinformatics Analysis We examined missense, nonsense, splice changes, and coding indels in the exome data that matched dominant or recessive inheritance models. The results were filtered to exclude variants with the following characteristics: synonymous variants, variants within known pseudogenes, variants with a minor allele frequency (MAF) greater than 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 the control group in our in-house exome variant database. We then referenced the Online Mendelian Inheritance in Man database to prioritize genes based on fulminant prediction and biological relevance.

[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 Medium supplemented with 10% fetal bovine serum. Primary adult HDLEC cells 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 number 23725)41 obtained from Addgene was amplified from the original vector and cloned into a pcDNA3.1 vector containing two copies of the FLAG tag (DDKDDK) followed by two STREP tags (WSHPQFEK) as BamHI / XhoI fragments. The S214P mutation was site-directed mutagenesis using NEB's Q5 mutagenesis kit according to the manufacturer's instructions. Transfection of HEK293T and HeLa cells was performed using Fugene HD (Promega) with 3 μg of DNA (empty vector, WT ARAF (ARAF-WT), or ARAF variant (ARAF-S214P)) and 9 μl of transfection reagent, according to the manufacturer's protocol. 36–48 hours after transfection, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice with freshly ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 100 mM NaCl, and 100 mM NaCl. Cells were lysated at a rate of 20 μl per ml of 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 a completely EDTA-free protease inhibitor cocktail (Roche Applied Science). After centrifugation to clear the cell lysates, the supernatant was collected and used for Western blotting or immunoprecipitation using Anti-FLAG M2 Affinity Gel (cat. no. A2220, Sigma), followed by Western blotting.Immunoprecipitation and lysates were analyzed using NuPAGE 4-12% Bis-Tris gel (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). Antibodies used included those from Cell Signaling Technology (1:1,000), 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 previously described pCDNA3.1-F2S2-ARAF-WT or -S214P construct was cleaved with BamHI / XhoI and introduced into the BglII / XhoI site of a modified pMSCV plasmid containing the amino-terminal FLAG and HA tag. Virus production was performed using Fugene, with 8 μg of total DNA (pMSCV-ARAF-WT or -S214P along with the envelope plasmid and packaging plasmid) and 18 μl of transfection reagent placed in a HEK293T. After 72 hours, the viral supernatant was collected and filtered. The cell culture medium was replaced with the viral supernatant, and 8 μg ml of transfection reagent was added. -1HDLEC was introduced by supplementing with polyblen and filtering through a 0.45 μm filter. Cells were spin-infected at 650 g for 90 minutes, followed by 6 hours of culture, at which point the viral supernatant was replaced with standard culture medium. Introduced HDLEC was cultured for 48 hours. Confirmation was performed before use in the experiment. The introduction efficiency observed by HA staining ranged from 40% to 60%.

[0172] Immunofluorescence staining and Western blotting of HDLECs Round (12 mm) coverslips (VWRs) were coated onto 24-well plates (Corning) with 0.1% gelatin dissolved in water for 10 minutes, then air-dried for 15 minutes. The introduced HDLECs were plated in 0.5 ml of culture medium at a rate of 100,000 cells per well and cultured for 48 hours in or without trametinib. The cells were washed with warm serum-free Dulbecco's modified Eagle medium and fixed with 4% paraformaldehyde. The fixed cells were washed twice with PBS and twice with 0.1% BSA in PBS. The cells were permeabilized and incubated with 10% normal donkey serum (Jackson Immunoresearch) and 0.3% Triton X-100 (Sigma Aldrich) in PBS and then blocked. Dilute VE-cadherin antibody (Thermo Fisher Scientific) with 0.01% normal donkey serum, 0.1% BSA, and 0.3% Triton X-100 (PBS) (final concentration: 2 μg ml). -1)The samples were then stained for 1 hour. The coverslips were washed twice with 0.1% BSA (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 hour. If 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 hour. The coverslips were washed twice with 0.1% BSA in PBS and twice with PBS. The coverslips were immersed in water to remove residual salt, and then mounted onto 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. The images were further processed with Fiji software package 42. Brightness and contrast adjustments were made. The same brightness and contrast settings were applied to all images. Fluorescence values ​​were measured in regions of interest (ROIs) drawn to include the entire cell, or ROIs drawn to include the entire cell body but not the cell-cell junctions. From these measurements, the cell membrane value (whole cell - intracellular) was calculated, and the ratio of the cell membrane to intracellular values ​​was calculated and plotted. Cell length and width were also measured using the line tool and ROI manager. In all analyses, five cells per 1 × 10⁻⁶ field were analyzed, based on HA staining indicating clear ARAF expression. Five × 10⁻⁶ fields were obtained for each condition in each experiment. The experiments were conducted using cells obtained from three independent thawing and HDLEC introduction processes, with a total of 75 cells used per condition.For Western blotting of HDLECs with trametinib, 20,000 transfused HDLECs cells were plated into 96-well plates in the presence of increasing amounts of trametinib. After culturing the cells in the presence of the drug for 24 hours, they were lysed with a cocktail of 40 mM HEPES pH 7.5, 120 mM NaCl, 0.3% CHAPS, 50 mM NaF, 1.5 mM NaVO3, and a protease inhibitor. The lysate was cleared by centrifugation at 20,000 g for 5 minutes at 4°C. Proteins were separated on a 4-12% NuPAGE Bis-Tris gel. Blotting was performed using the antibodies described above.

[0173] 3D lymphocyte spheroid germination assay Multicellular spheroids for lymphatic vessel sprouting assays were prepared by seeding 7,500 HDLEC cells expressing ARAF-WT or ARAF-S214P into the wells of a 96-well plate pre-coated with 1.5% agarose. Under these conditions, all HDLEC cells aggregated into a single spheroid within 24 hours. After formation, each spheroid was transferred to a gelling solution consisting of type I collagen (cat No. 354236 Corning; final concentration). The spheroids were then transferred to a gelling solution containing trametinib at the indicated concentration and polymerized at 37°C. Once solidified, Endothelial Cell Growth Medium MV2 (without VEGFC) containing an appropriate concentration of trametinib was added to the collagen gel. After two days of incubation, Z-stack images of the embedded spheroids were acquired using the EVOS FL Auto Imaging System (Thermo Fisher Scientific) with a step size of 8.5 μm. The number and length of capillary buds growing from each spheroid were measured using ImageJ software (https: / / imagej.nih.gov / ij / ).

[0174] MTT proliferation assay using transformed HDLEC The growth of introduced HDLEC cells was measured using Roche Applied Science's Cell Proliferation Kit I (MTT). Briefly, on day 2 after retrovirus introduction, ARAF-WT- and -S214P-expressing HDLEC cells were collected, counted, and replicated in 100 μl of medium at 10,000 cells per well in a flat-bottomed 96-well plate. At the time indicated after plating, 10 μl of MTT was added to the appropriate wells and incubated at 37°C for 4 hours. After adding 100 μl of solubilizing reagent, the cells were incubated overnight at 37°C. Absorbance at 550 nm and 700 nm was measured using a Spectramax i3 Multi Mode plate reader (Molecular Devices), and A550 nm - A700 nm values ​​were calculated. The 4-hour mark after plating was included as a guideline for when cells with low growth were introduced into the experiment.

[0175] Transgenic expression of human ARAF in zebrafish All procedures using zebrafish were approved by the CHOP's Institutional Animal Care and Use Committee (IAC 001154) and followed the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Human variant and WT ARAF cDNA were cloned into the pDONR221 vector without stop codons; a zebrafish-adapted Kozak sequence (GCAAACATGG) was used.43 Expression constructs were assembled using a Tol2 backbone vector containing a gateway cloning cassette.44,45 Tol2 messenger RNA was co-injected into the constructs. (46)

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

[0177] To analyze TD dilation, somites separated by intersegmental lymphatic vessels expressing transgenes in TD were selected. Morphology was evaluated as follows: normal (WT), moderate dilation (TD is dilated but separate from PCV), or severe dilation (TD and PCV are indistinguishable by Z projection). Images were edited using ImageJ (Fiji). Each experiment was performed three times, and a total of 40 animals were analyzed.

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

[0179] Staining of zebrafish with p-ERK antibody Fish were injected as described above, and larvae exhibiting significant expression of WT or mutant ARAF / mcherry were selected for analysis. Larvae were fixed overnight in a 4% paraformaldehyde solution in PBS (PBST) containing Tween-20. The larvae were washed with PBST and incubated for 24 hours at 4°C in 2% Triton X-100. Subsequently, the larvae were blocked with 10% bovine serum and incubated overnight at 4°C. They were stained with phospho-ERK T202 / Y204 antibody (cat.no.9101, Cell Signaling Technology, 1:200), washed with PBST, and then 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-sided Student's t-tests to compare the two groups. Statistical analysis was performed using GraphPad Prism 7.0d software. Data were presented as dot plots using boxes at the 25th–75th percentiles, whiskers from minimum to maximum, box and whisker plots centered on the median, or bar graphs with mean ± sem. For all assays, except for proliferation assays where statistical analysis was not performed, three independent experiments were conducted with independent transfection of HDLEC cells. For the 14-3-3 protein association assay, three independent experiments were conducted with independent transfection of HEK293T cells, while other results for HEK293T cells represent six independent experiments. All zebrafish-related assays were conducted with three independent experiments, and the following unlogarithmic one-sided Student's t-tests were used to compare the two groups.

[0181] result The first stage of WES analysis of known lymphoid abnormality-associated genes, including mutation analysis of AKT1, PIK3CA, KRAS, HRAS, NRAS, BRAF, RAF1, PTPN11, SHOC2, CBL, RIT1, and SOS1, did not reveal any significant abnormalities. Subsequently, prioritizing the genes revealed a novel mutation in ARAF on the X chromosome, c.640T>C:p.S214P. Patient P1 was male and had developed CCLA (Figure 22A, 22C-22E; see "Methods" for detailed clinical symptoms, GenBank Accession No. NG_016339), while patient P2 was female and had a history of lymphangiomatosis. This mutation affected a conserved phosphorylation site, and since residue 214 is a paralogous regulatory site for repression by the 14-3-3 protein in the homologous protein C-RAF (also known as RAF1), it is likely that a gain-of-function (GoF) effect was achieved. 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 internal database. Sanger sequencing of blood-derived Sanger sequencing of P1 and DNA from both parents confirmed that this X-linked ARAF mutation occurred in a somatic heterozygous state, similar to male patients (Fig. 22F). Sanger sequencing of ARAF mutations in P2, his unaffected daughter, and his mother confirmed that the mutation was present only in P2 (Fig. 22F). The father was unable to participate in sequencing, but since he did not report any respiratory symptoms, it is likely that the ARAF mutation in P2 occurred as a de novo or somatic mutation. Patient P2 could not be followed up and was later informed that he died five years after diagnosis from complications of lymphatic disease.

[0182] Ser 214, one of the 14-3-3 binding sites of ARAF, is located in the conserved region 2 (CR2)(14) and, like the RAF protein, is highly conserved across vertebrate species, suggesting it plays an essential role in the function of these kinases (Figure 22G). It is thought that the 14-3-3 protein can prevent the recruitment of the ARAF protein to the cell membrane by activated Ras by binding to the phosphorylated cell 214 of ARAF (15). Previous studies have shown that a mutation at the ARAF-S214-like residue 259 of such C-RAF inhibits binding to the 14-3-3 protein, leading to localization to the cell membrane and induction of ERK / MEK signaling (16). As shown in Figure 23A, HEK293T cells transfected with ARAF-S214P showed reduced co-precipitation of the 14-3-3 protein and significantly greater ERK1 / 2 activation, as measured by increased phosphorylation, compared to 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 and growth factors.

[0183] HDLECs expressing ARAF-S214P showed improved lymphatic vessel formation compared to HDLECs expressing ARAF-WT, and both the number and length of spheroids were measured in a 3D lymphocyte spheroid sprouting test conducted in the absence of vascular endothelial growth factor C (VEGFC) (Fig. 23D). The MEK inhibitor trametinib restored the increase in mutant sprouting (Fig. 23D). Next, morphological analysis of the adrenal junctions of the endothelium of primary HDLECs expressing ARAF-S214P was performed. Immunofluorescence microscopy revealed that when ARAF-S214P was expressed, the accumulation of VE-cadherin between adjacent cells was significantly reduced, suggesting that VE-cadherin internalization was promoted (Fig. 23E, yellow arrowheads). Furthermore, expressed ARAF-S214P altered the structure of actin, and the number of F-actin filaments in cells expressing the mutant was reduced.

[0184] Next, we investigated the ability of MEK1 / 2 inhibitors to restore these abnormalities. Administration of the MEK inhibitor trametinib at a concentration of 100 nM restored the loss of VE-cadherin from intercellular junctions observed in HDLECs expressing ARAF-S214P, nearly completely restored cellular monolayerity, and restored normal appearance of VE-cadherin in junctions and actin filaments (Figure 23E). Although ARAF-S214P is clearly activated, ERK is activated in HDLECs, and ERK activation is generally associated with cell proliferation; however, no measurable difference in proliferation was observed between ARAF-WT and cells expressing -S214P. HDLECs were detected with two independent retroviral transductions (Figure 23F).

[0185] Lymphatic vessel development in zebrafish was analyzed using the Tg(mrc1a:egfp)y251 transgenic line 17. In this transgenic line, all lymphatic endothelial cells were labeled with EGFP. ARAF expression was targeted to lymphatic vessels using the mrc1a promoter, and cells expressing ARAF were indicated by mCherry expression. Expression of ARAF-S214P induced lymphatic vessel dilation at various locations, with the most consistent observation being dilation of the stem TD (Fig. 23G). On the other hand, expression of ARAF-WT also affected lymphatic vessel morphology (Fig. 23H). ARAF-S214P expression induces p-ERK in zebrafish. To investigate whether the abnormality can be reversed with MEK signaling inhibitors, mrc1a:ARAFS214P larvae were treated with cobimetinib from day 3 postfertilization (dpf), when lymphoid progenitor cells sprout and TDs form (17). Analysis of somites revealed that ARAF expression was significantly improved by cobimetinib, resulting in improved duct morphology (Figures 23I, 23J). On the other hand, WT Tg(mrc1a:egfp)y251 larvae were well tolerated by the drug when administered cobimetinib.

[0186] Based on our findings that ARAF mutations lead to P1 gain-of-function and non-response to sirolimus, and that MEK inhibitors rescue the lymphoid phenotype in endothelial cells and transgenic zebrafish models, we hypothesized the following:

[0187] Institutional Review Board approval was obtained to use MEK inhibitor therapy in Phase 1. Subsequently, trametinib (Mekinist), a MEK inhibitor approved by the U.S. Food and Drug Administration (FDA), was used off-label in this 12-year-old patient after a comprehensive baseline evaluation.

[0188] The starting dose of trametinib is 1 mg d -1Within two months of the start of treatment, improvements began to be seen in pulmonary function tests (Figure 24A). Furthermore, three months after the start of treatment, lymphatic fluid retention and oxygen supply decreased significantly, the level of physical activity improved, and the patient was able to transition to room air without experiencing any adverse events from trametinib. Pulmonary function tests 12 months after the start of treatment showed that total vital capacity (TLC) had almost doubled, and forced expiratory volume in one second (FEV1) improved from 23% of the predicted value to 42% (Figure 24A). Electrolytes (low Na, low K) normalized, and magnetic resonance imaging scans showed lymphatic system reconstruction (Figures 24B-24F), with significant recovery including a doubling of total vital capacity (TLC) and forced expiratory volume (Figure 24C). Volume in one second (FEV1) improved from 23% of the predicted value to 42% (Figure 24A). Electrolytes (low Na, low K) normalized, and magnetic resonance imaging scans confirmed lymphatic system reconstruction (Figures 24B-24F). This patient, who had been frequently hospitalized before starting this gene induction therapy, made a remarkable recovery (Figure 24F).

[0189] As described above, we performed WES on two unrelated patients with lymphatic vessel abnormalities and confirmed recurrent gain-of-function mutations in the ARAF gene, including a 12-year-old male with advanced lymphatic vessel disease that did not respond to sirolimus therapy. When the HDLECs mutant ARAF was introduced, we observed increased ERK1 / 2 activity, enhanced lymphangiogenesis, and disruption of the actin cytoskeleton and VE-cadherin junctions, but these conditions were reversed.

[0190] This was performed using the MEK inhibitor trametinib. In HDLEC cells expressing ARAF-S214P, sprouting was observed in the absence of VEGFC (potent lymphangiogenic factor) (18). Under the same conditions, no sprouting was observed in cells expressing ARAF-WT. From this, it is thought that ARAF mutants mimic the stimulation behavior of VEGFC, or that HDLEC induces the expression of VEGFC, which is necessary for endothelial cell sprouting, as is seen in many stromal cells (19-21). We reproduced the abnormal lymphoid phenotype. This was done by observing the recovery of the phenotype with a MEK inhibitor using a zebrafish model of patients with GoFARAF mutations. Surprisingly, when trametinib was administered to proband patients with ARAF gene mutations, the patients' symptoms dramatically improved, and within 12 months of the start of treatment, the dilated and turbulent lymphatic vessels were reconstructed, lymphedema was resolved, and they were able to resume normal daily life.

[0191] In our ongoing patient recruitment, we investigated patients with even more lymphatic abnormalities, including Noonan (or Noonan-related) syndrome, Gorham-Stout disease, lymphangiomatosis scapularis (KLA), lymphangiectasia, and CCLA. Further sequencing of 43 patients revealed seven additional mutations in KRAS, BRAF, RASA1, PTPN11, and SOS1 (Table 3). This suggests that the RAS-MAPK signaling pathway is a common pathway causing the clinical symptoms of various lymphatic disorders. Indeed, the RAS-MAPK pathway is increasingly recognized as playing a crucial role in lymphangiogenesis signaling (21-23). ​​We identified more than 50 patients with mutations in KRAS, HRAS, BRAF, RAF1, PTPN11, SHOC2, CBL, RIT1, and SOS1, exhibiting clinical features of Noonan syndrome or Noonan-related syndrome, accompanied by lymphatic abnormalities such as pleural effusion, pericardial effusion, pneumothorax, and edema. During our research, it became clear that recurrent NRAS mutations are involved in GLA(37) and KLA(38), further supporting the common genetic etiology of these disease groups and the importance of mutations in the RAS-MAPK pathway in lymphatic abnormalities.

[0192] The widespread presence of RASopathies mutations in human cancers has been recognized for decades. When we examined the ARAF mutation we discovered using the cBioPortal (39) database (n=71,857 subjects, accessed February 6, 2019), we found two patients with the exact same ARAF mutation. Interestingly, both patients also had the TP53 mutation, which is considered a carcinogenic driver. Furthermore, three of the different mutations at this residue (S214T, S214A, S214Y, S214C, S214F) have been shown to increase MEK / ERK phosphorylation (40), and these were observed in 10 patients with different types of cancer. However, in 9 of these 10 patients, oncogenic mutations coexisted, specifically TP53, GNAS, AKT2, APC, EGFR, ATM, CHEK2, KIT, and U2AF1, suggesting that these carcinogenic drivers may be responsible for the excessive proliferation of cancer cells. Primary screening patients with ARAF mutations have dilated lymphatic vessels, but their lesion size has not increased over years of follow-up. Therefore, these data are consistent with our observation that the ARAF mutation we discovered may not promote lymphatic vessel proliferation.

[0193] Endothelial cells in vitro Regarding the prevalence of mutation-positive lymphangiomalgias, among the 11 US centers that have formed the Lymphangiomalgias Consortium to facilitate multicenter clinical trials of this group of lymphangiomalgias, which includes but is not limited to GLA, Gorham-Stout disease, CCLA, KLA, Klippel-Trenaunay syndrome, and kaposiform hemangioendothelioma, more than 3,000 patients with moderate to severe disease progression have been recruited, with a combined annual number of approximately 300 new patients. Based on the current molecular diagnostic yield (20%), it is estimated that approximately 20% of these have RAS-MAPK pathway deficiencies, suggesting that thousands of patients across the US could benefit from treatment with MEK inhibitors. Thus, our study illustrates how gene discovery can impact disease classification and lead to the discovery of new biological and life-saving therapies, enabling previously unknown etiologies in patients and facilitating precision medicine approaches.

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[0195] While some preferred embodiments of the present invention have been described and illustrated above, the present invention is not intended to be limited to these embodiments. Various modifications can be made without departing from the scope and spirit of the invention, as described in the following claims.

Claims

1. The use of one or more agents in the manufacture of drugs for the treatment of central duct lymphatic disorder (CCLA) in human patients, (a) (i) KRAS c. 35G>A: pG12D, (ii) BRAF c. 1403T>C:pF468S and c. 2128-G>T, (iii) SOS1 c. 2536G>A: pE846K, (iv) ITGA9 c. 1236+4A>G and c. 289T>G: p. C97G, (v) c. 475_476del:p. (L159Gfs * 20) and c. 2246G>Cp. R749P of RASA1, (vi) RAF1 c. 433A > C:p. T145P, (vii) RIT1 c. 270G>T:p. M90I, (viii) PEIZO1 c. 7289C>T:p. P2430L, (ix) NF1 c. 1034_1043del:p. (L345Pfs * 28), and (x) CBL c. 1096-1G>T and c. 2322T>G: p. Y774 * One or more single nucleotide variants (SNVs) are detected in the nucleic acids of the biological sample obtained from the patient, and, (b) Each of the drugs is independently trametinib (GSK1120212), rapamycin (sirolimus), everolimus (RAD001), AZD8055, temsirolimus (CCI-779, NSC 683864), KU-0063794, MHY1485, BEZ235 (NVP-BEZ235, dactricib), PI-103, tolkinib (PP242), tacrolimus (FK506), selumetinib (AZD6244), PD0325901, PD184352 (CI-1040), pimacertib (AS-703026), TAK-733, AZD8330, Binimetinib (MEK162, ARRY-162, ARRY-438162), SL-327, Rifametinib (RDEA119, Bay86-9766), Cobimetinib (GDC-0973, RG7420), Urixertinib, Ridaforolimus (Deforolimus, MK-8669), INK 128 (MLN0128), voxatalisib (SAR245409, XL765), torin1, omiparicib (GSK2126458, GSK458), OSI-027, PF-04691502, apitricib (GDC-0980, RG7422), GSK1059615, jedatlicib (PF-05212384, PKI-587), WYE-354, AZD2014, torin2, WYE-125132 (WYE-132), P The following are 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), paromide 529 (P529), chrysophanic acid, TAK-733, PD-325901, pimacertib (AS-703026), PD184352, and SL-327. use.

2. The use according to claim 1, wherein the lymphatic disorder is characterized by abnormal formation of lymphatic vessels and / or tissue overgrowth.

3. The use according to any one of claims 1 to 2, wherein the lymphatic abnormality is characterized by chyle exudate containing pericardial fluid, pleural fluid, or peritoneal fluid.

4. The use according to claim 1, wherein the report identifying the SNV is generated after detection in the biological sample.

5. The use according to any one of claims 1 to 4, wherein the drug is at least one selected from the group consisting of trametinib, pimacertib, and rifametinib.

6. Use according to any one of claims 1 to 5, wherein the patient does not have one SNV selected from the group consisting of PTPN11, KRAS, BRAF, SOS1, and ITGA9.

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

8. The use according to any one of claims 1 to 7, wherein the biological sample comprises DNA or RNA.

9. The use according to any one of claims 1 to 8, wherein the nucleic acid comprising the SNV is obtained from isolated cells of a human patient.

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