Delineation of LZTR1 mutation-positive patients with Noonan syndrome and identification of LZTR1 binding to RAF1–PPP1CB complexes
RASopathies are a group of developmental disorders caused by mutations in genes that regulate the RAS/MAPK pathway and include Noonan syndrome (NS), Costello syndrome, cardiofaciocutaneous syndrome and other related disorders. Whole exome sequencing studies recently identified LZTR1, PPP1CB and MRAS as new causative genes in RASopathies. However, information on the phenotypes of LZTR1 mutation-positive patients and functional properties of the mutations are limited. To identify variants of LZTR1, PPP1CB, and MRAS, we performed a targeted next-generation sequencing and reexamined previously analyzed exome data in 166 patients with suspected RASopathies. We identified eight LZTR1 variants, including a de novo variant, in seven probands who were suspicious for NS and one known de novo PPP1CB variant in a patient with NS. One of the seven probands had two compound heterozygous LZTR1 variants, suggesting autosomal recessive inheritance. All probands with LZTR1 variants had cardiac defects, including hypertrophic cardiomyopathy and atrial septal defect. Five of the seven probands had short stature or intellectual disabilities. Immunoprecipitation of endogenous LZTR1 followed by western blotting showed that LZTR1 bound to the RAF1–PPP1CB complex. Cells transfected with a small interfering RNA against LZTR1 exhibited decreased levels of RAF1 phosphorylated at Ser259. These are the first results to demonstrate LZTR1 in association with the RAF1–PPP1CB complex as a component of the RAS/MAPK pathway.
The authors thank the patients, their family members, and the doctors who participated in this study. We are grateful to Jun-ichi Miyazaki of Osaka University for supplying the pCAGGS expression vector. We thank Daiju Oba, Ayumi Nishiyama, Shingo Takahara, Aya Shibui-Inoue, Yu Katata and Koki Nagai who contributed to the routine diagnostic work, and Yoko Tateda, Kumi Kato, and Riyo Takahashi for their technical assistance.
This study was supported in part by the Grants-in-Aid by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, AMED to Y.A. (18ek0109241h0002), and the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 17H04223 to Y.A. and 18K15657 to T.A.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Aoki Y, Niihori T, Banjo T, Okamoto N, Mizuno S, Kurosawa K, Ogata T, Takada F, Yano M, Ando T, Hoshika T, Barnett C, Ohashi H, Kawame H, Hasegawa T, Okutani T, Nagashima T, Hasegawa S, Funayama R, Nagashima T, Nakayama K, Inoue S, Watanabe Y, Ogura T, Matsubara Y (2013) Gain-of-function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome. Am J Hum Genet 93:173–180. https://doi.org/10.1016/j.ajhg.2013.05.021 CrossRefGoogle Scholar
- Chen PC, Yin J, Yu HW, Yuan T, Fernandez M, Yung CK, Trinh QM, Peltekova VD, Reid JG, Tworog-Dube E, Morgan MB, Muzny DM, Stein L, McPherson JD, Roberts AE, Gibbs RA, Neel BG, Kucherlapati R (2014) Next-generation sequencing identifies rare variants associated with Noonan syndrome. Proc Natl Acad Sci USA 111:11473–11478. https://doi.org/10.1073/pnas.1324128111 CrossRefGoogle Scholar
- Farschtschi S, Mautner VF, Pham M, Nguyen R, Kehrer-Sawatzki H, Hutter S, Friedrich RE, Schulz A, Morrison H, Jones DT, Bendszus M, Baumer P (2016) Multifocal nerve lesions and LZTR1 germline mutations in segmental schwannomatosis. Ann Neurol 80:625–628. https://doi.org/10.1002/ana.24753 CrossRefGoogle Scholar
- Frattini V, Trifonov V, Chan JM, Castano A, Lia M, Abate F, Keir ST, Ji AX, Zoppoli P, Niola F, Danussi C, Dolgalev I, Porrati P, Pellegatta S, Heguy A, Gupta G, Pisapia DJ, Canoll P, Bruce JN, McLendon RE, Yan H, Aldape K, Finocchiaro G, Mikkelsen T, Prive GG, Bigner DD, Lasorella A, Rabadan R, Iavarone A (2013) The integrated landscape of driver genomic alterations in glioblastoma. Nat Genet 45:1141–1149. https://doi.org/10.1038/ng.2734 CrossRefGoogle Scholar
- Gripp KW, Aldinger KA, Bennett JT, Baker L, Tusi J, Powell-Hamilton N, Stabley D, Sol-Church K, Timms AE, Dobyns WB (2016) A novel rasopathy caused by recurrent de novo missense mutations in PPP1CB closely resembles Noonan syndrome with loose anagen hair. Am J Med Genet A 170:2237–2247. https://doi.org/10.1002/ajmg.a.37781 CrossRefGoogle Scholar
- Hutter S, Piro RM, Reuss DE, Hovestadt V, Sahm F, Farschtschi S, Kehrer-Sawatzki H, Wolf S, Lichter P, von Deimling A, Schuhmann MU, Pfister SM, Jones DTW, Mautner VF (2014) Whole exome sequencing reveals that the majority of schwannomatosis cases remain unexplained after excluding SMARCB1 and LZTR1 germline variants. Acta Neuropathol 128:449–452. https://doi.org/10.1007/s00401-014-1311-1 CrossRefGoogle Scholar
- Johnston JJ, van der Smagt JJ, Rosenfeld JA, Pagnamenta AT, Alswaid A, Baker EH, Blair E, Borck G, Brinkmann J, Craigen W, Dung VC, Emrick L, Everman DB, van Gassen KL, Gulsuner S, Harr MH, Jain M, Kuechler A, Leppig KA, McDonald-McGinn DM, Can NTB, Peleg A, Roeder ER, Rogers RC, Sagi-Dain L, Sapp JC, Schaffer AA, Schanze D, Stewart H, Taylor JC, Verbeek NE, Walkiewicz MA, Zackai EH, Zweier C, Zenker M, Lee B, Biesecker LG (2018) Autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants. Genet Med. https://doi.org/10.1038/gim.2017.249 Google Scholar
- Kobayashi T, Aoki Y, Niihori T, Cave H, Verloes A, Okamoto N, Kawame H, Fujiwara I, Takada F, Ohata T, Sakazume S, Ando T, Nakagawa N, Lapunzina P, Meneses AG, Gillessen-Kaesbach G, Wieczorek D, Kurosawa K, Mizuno S, Ohashi H, David A, Philip N, Guliyeva A, Narumi Y, Kure S, Tsuchiya S, Matsubara Y (2010) Molecular and clinical analysis of RAF1 in Noonan syndrome and related disorders: dephosphorylation of serine 259 as the essential mechanism for mutant activation. Hum Mutat 31:284–294. https://doi.org/10.1002/humu.21187 CrossRefGoogle Scholar
- Ma L, Bayram Y, McLaughlin HM, Cho MT, Krokosky A, Turner CE, Lindstrom K, Bupp CP, Mayberry K, Mu W, Bodurtha J, Weinstein V, Zadeh N, Alcaraz W, Powis Z, Shao Y, Scott DA, Lewis AM, White JJ, Jhangiani SN, Gulec EY, Lalani SR, Lupski JR, Retterer K, Schnur RE, Wentzensen IM, Bale S, Chung WK (2016) De novo missense variants in PPP1CB are associated with intellectual disability and congenital heart disease. Hum Genet 135:1399–1409. https://doi.org/10.1007/s00439-016-1731-1 CrossRefGoogle Scholar
- Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, Niihori T, Aoki Y, Matsubara Y, Arveiler B, Lacombe D, Pasmant E, Parfait B, Baumann C, Heron D, Sigaudy S, Toutain A, Rio M, Goldenberg A, Leheup B, Verloes A, Cave H (2007) Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype-phenotype relationships and overlap with Costello syndrome. J Med Genet 44:763–771. https://doi.org/10.1136/jmg.2007.050450 CrossRefGoogle Scholar
- Nishiyama A, Niihori T, Warita H, Izumi R, Akiyama T, Kato M, Suzuki N, Aoki Y, Aoki M (2017) Comprehensive targeted next-generation sequencing in Japanese familial amyotrophic lateral sclerosis. Neurobiol Aging 53:194.e1–194.e194. https://doi.org/10.1016/j.neurobiolaging.2017.01.004 e8.CrossRefGoogle Scholar
- Piotrowski A, Xie J, Liu YF, Poplawski AB, Gomes AR, Madanecki P, Fu C, Crowley MR, Crossman DK, Armstrong L, Babovic-Vuksanovic D, Bergner A, Blakeley JO, Blumenthal AL, Daniels MS, Feit H, Gardner K, Hurst S, Kobelka C, Lee C, Nagy R, Rauen KA, Slopis JM, Suwannarat P, Westman JA, Zanko A, Korf BR, Messiaen LM (2013) Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas. Nat Genet 46:182–187. https://doi.org/10.1038/ng.2855 CrossRefGoogle Scholar
- Rodriguez-Viciana P, Oses-Prieto J, Burlingame A, Fried M, McCormick F (2006) A phosphatase holoenzyme comprised of Shoc2/Sur8 and the catalytic subunit of PP1 functions as an M-Ras effector to modulate Raf activity. Mol Cell 22:217–230. https://doi.org/10.1016/j.molcel.2006.03.027 CrossRefGoogle Scholar
- Smith MJ, Isidor B, Beetz C, Williams SG, Bhaskar SS, Richer W, O’Sullivan J, Anderson B, Daly SB, Urquhart JE, Fryer A, Rustad CF, Mills SJ, Samii A, du Plessis D, Halliday D, Barbarot S, Bourdeaut F, Newman WG, Evans DG (2015) Mutations in LZTR1 add to the complex heterogeneity of schwannomatosis. Neurology 84:141–147. https://doi.org/10.1212/WNL.0000000000001129 CrossRefGoogle Scholar
- Villani A, Greer MC, Kalish JM, Nakagawara A, Nathanson KL, Pajtler KW, Pfister SM, Walsh MF, Wasserman JD, Zelley K, Kratz CP (2017) Recommendations for cancer surveillance in individuals with RASopathies and other rare genetic conditions with increased cancer risk. Clin Cancer Res 23:e83–e90. https://doi.org/10.1158/1078-0432.CCR-17-0631 CrossRefGoogle Scholar
- Vissers LE, Bonetti M, Paardekooper Overman J, Nillesen WM, Frints SG, de Ligt J, Zampino G, Justino A, Machado JC, Schepens M, Brunner HG, Veltman JA, Scheffer H, Gros P, Costa JL, Tartaglia M, van der Burgt I, Yntema HG, den Hertog J (2015) Heterozygous germline mutations in A2ML1 are associated with a disorder clinically related to Noonan syndrome. Eur J Hum Genet 23:317–324. https://doi.org/10.1038/ejhg.2014.115 CrossRefGoogle Scholar
- Yamamoto GL, Aguena M, Gos M, Hung C, Pilch J, Fahiminiya S, Abramowicz A, Cristian I, Buscarilli M, Naslavsky MS, Malaquias AC, Zatz M, Bodamer O, Majewski J, Jorge AA, Pereira AC, Kim CA, Passos-Bueno MR, Bertola DR (2015) Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome. J Med Genet 52:413–421. https://doi.org/10.1136/jmedgenet-2015-103018 CrossRefGoogle Scholar
- Yaoita M, Niihori T, Mizuno S, Okamoto N, Hayashi S, Watanabe A, Yokozawa M, Suzumura H, Nakahara A, Nakano Y, Hokosaki T, Ohmori A, Sawada H, Migita O, Mima A, Lapunzina P, Santos-Simarro F, Garcia-Minaur S, Ogata T, Kawame H, Kurosawa K, Ohashi H, Inoue SI, Matsubara Y, Kure S, Aoki Y (2016) Spectrum of mutations and genotype–phenotype analysis in Noonan syndrome patients with RIT1 mutations. Hum Genet 135:209–222. https://doi.org/10.1007/s00439-015-1627-5 CrossRefGoogle Scholar
- Young LC, Hartig N, Munoz-Alegre M, Oses-Prieto JA, Durdu S, Bender S, Vijayakumar V, Vietri Rudan M, Gewinner C, Henderson S, Jathoul AP, Ghatrora R, Lythgoe MF, Burlingame AL, Rodriguez-Viciana P (2013) An MRAS, SHOC2, and SCRIB complex coordinates ERK pathway activation with polarity and tumorigenic growth. Mol Cell 52:679–692. https://doi.org/10.1016/j.molcel.2013.10.004 CrossRefGoogle Scholar