Microphthalmia-associated transcription factor
|, CMM8, MI, WS2, WS2A, bHLHe32, microphthalmia-associated transcription factor, melanogenesis associated transcription factor, COMMAD, melanocyte inducing transcription factor|
MITF is a basic helix-loop-helix leucine zipper transcription factor involved in lineage-specific pathway regulation of many types of cells including melanocytes, osteoclasts, and mast cells. The term "lineage-specific", since it relates to MITF, means genes or traits that are only found in a certain cell type. Therefore, MITF may be involved in the rewiring of signaling cascades that are specifically required for the survival and physiological function of their normal cell precursors.
MITF is the most characterized member of the MIT family. Its gene resides at the mi locus in mice, and its protumorogenic targets include factors involved in cell death, DNA replication, repair, mitosis, microRNA production, membrane trafficking, mitochondrial metabolism, and much more. Mutation of this gene results in deafness, bone loss, small eyes, and poorly pigmented eyes and skin. In human subjects, because it is known that MITF controls the expression of various genes that are essential for normal melanin synthesis in melanocytes, mutations of MITF can lead to diseases such as melanoma, Waardenburg syndrome, and Tietz syndrome. Its function is conserved across vertebrates, including in fishes such as zebrafish and Xiphophorus.
An understanding of MITF is necessary to understand how certain lineage-specific cancers and other diseases progress. In addition, current and future research can lead to potential avenues to target this transcription factor mechanism for cancer prevention.
Waardenburg syndrome is a rare genetic disorder. Its symptoms include deafness, minor defects, and abnormalities in pigmentation. Mutations in the MITF gene have been found in certain patients with Waardenburg syndrome, type II. Mutations that change the amino acid sequence of that result in an abnormally small MITF are found. These mutations disrupt dimer formation, and as a result cause insufficient development of melanocytes. The shortage of melanocytes causes some of the characteristic features of Waardenburg syndrome.
Tietz syndrome, first described in 1923, is a congenital disorder often characterized by deafness and leucism. Tietz is caused by a mutation in the MITF gene. The mutation in MITF deletes or changes a single amino acid base pair specifically in the base motif region of the MITF protein. The new MITF protein is unable to bind to DNA and melanocyte development and subsequently melanin production is altered. A reduced number of melanocytes can lead to hearing loss, and decreased melanin production can account for the light skin and hair color that make Tietz syndrome so noticeable.
Melanocytes are commonly known as cells that are responsible for producing the pigment melanin which gives coloration to the hair, skin, and nails. The exact mechanisms of how exactly melanocytes become cancerous are relatively unclear, but there is ongoing research to gain more information about the process. For example, it has been uncovered that the DNA of certain genes is often damaged in melanoma cells, most likely as a result of damage from UV radiation, and in turn increases the likelihood of developing melanoma. Specifically, it has been found that a large percentage of melanomas have mutations in the B-RAF gene which leads to melanoma by causing an MEK-ERK kinase cascade when activated. In addition to B-RAF, MITF is also known to play a crucial role in melanoma progression. Since it is a transcription factor that is involved in the regulation of genes related to invasiveness, migration, and metastasis, it can play a role in the progression of melanoma. Figure 1 shows the specific activators and targets of MITF that are related to the survival, migration, proliferation, invasion and metastasis of melanoma cells.
MITF recognizes E-box (CAYRTG) and M-box (TCAYRTG or CAYRTGA) sequences in the promoter regions of target genes. Known target genes (confirmed by at least two independent sources) of this transcription factor include,
The LysRS-Ap4A-MITF signaling pathway
The LysRS-Ap4A-MITF signaling pathway was first discovered in mast cells, in which, the MAPK pathway is activated upon allergen stimulation. Lysyl-tRNA synthetase (LysRS), which normally resides in the multisynthetase complex with other tRNA sythetases, is phosphorylated on Serine 207 in a MAPK-dependent manner. This phosphorylation causes LysRS to change its conformation, detach from the complex and translocate into the nucleus, where it associates with the MITF-HINT1 inhibitory complex. The conformational change switches LysRS activity from aminoacylation of Lysine tRNA to diadenosine tetraphosphate (Ap4A) production. Ap4A binds to HINT1, which releases MITF from the inhibitory complex, allowing it to transcribe its target genes. Activation of the LysRS-Ap4A-MITF signaling pathway by isoproterenol has been confirmed in cardiomyocytes, where MITF is a major regulator of cardiac growth and hypertrophy.
MITF is phosphorylated on several serine and tyrosine residues. Serine phosphorylation is regulated by several signaling pathways including MAPK/BRAF/ERK, receptor tyrosine kinase KIT and GSK-3. In addition, several kinases including PI3K, AKT, SRC and P38 are also critical activators of MITF phosphorylation. In contrast, tyrosine phosphorylation is induced by the presence of the KIT oncogenic mutation D816V. This KITD816V pathway is dependent on SRC protein family activation signaling. The induction of serine phosphorylation by the frequently altered MAPK/BRAF pathway and the GSK-3 pathway in melanoma regulates MITF nuclear export and thereby decreasing MITF activity in the nucleus. Similarly, tyrosine phosphorylation mediated by the presence of the KIT oncogenic mutation D816V also increases the presence of MITF in the cytoplasm.
Most transcription factors function in cooperation with other factors by protein–protein interactions. Association of MITF with other proteins is a critical step in the regulation of MITF-mediated transcriptional activity. Some commonly studied MITF interactions include those with MAZR, PIAS3, Tfe3, hUBC9, PKC1, and LEF1. Looking at the variety of structures gives insight into MITF's varied roles in the cell.
The Myc-associated zinc-finger protein related factor (MAZR) interacts with the Zip domain of MITF. When expressed together, both MAZR and MITF increase promoter activity of the mMCP-6 gene. MAZR and MITF together transactivate the mMCP-6 gene. MAZR also plays a role in the phenotypic expression of mast cells in association with MITF.
PIAS3 is a transcriptional inhibiter that acts by inhibiting STAT3's DNA binding activity. PIAS3 directly interacts with MITF, and STAT3 does not interfere with the interaction between PIAS3 and MITF. PIAS3 functions as a key molecule in suppressing the transcriptional activity of MITF. This is important when considering mast cell and melanocyte development.
MITF and TFE3 are both part of the basic helix-loop-helix-leucine zipper family of transcription factors. Each protein encoded by the family of transcription factors can bind DNA. MITF is necessary for melanocyte and eye development, and new research suggests that TFE3 is also required for osteoclast development, a function redundant of MITF. The combined loss of both genes results in severe osteopetrosis, pointing to an interaction between MITF and other members of its transcription factor family.
UBC9 is a ubiquitin conjugating enzyme whose proteins associates with MITF. Although hUBC9 is known to act preferentially with SENTRIN/SUMO1, an in vitro analysis demonstrated greater actual association with MITF. hUBC9 is a critical regulator of melanocyte differentiation. To do this, it targets MITF for proteasome degradation.
Protein kinase C-interacting protein 1 (PKC1) associates with MITF. Their association is reduced upon cell activation. When this happens MITF disengages from PKC1. PKC1 by itself, found in the cytosol and nucleus, has no known physiological function. It does, however, have the ability to suppress MITF transcriptional activity and can function as an in vivo negative regulator of MITF induced transcriptional activity.
The functional cooperation between MITF and the lymphoid enhancing factor (LEF-1) results in a synergistic transactivation of the dopachrome tautomerase gene promoter, which is an early melanoblast marker. LEF-1 is involved in the process of regulation by Wnt signaling. LEF-1 also cooperates with MITF-related proteins like TFE3. MITF is a modulator of LEF-1, and this regulation ensures efficient propagation of Wnt signals in many cells.
Translational regulation of MITF is still an unexplored area with only two peer-reviewed papers (as of 2019) highlighting the importance. During glutamine starvation of melanoma cells ATF4 transcripts increases as well as the translation of the mRNA due to eIF2α phosphorylation. This chain of molecular events leads to two levels of MITF suppresssion: first, ATF4 protein binds and suppresses MITF transcription and second, eIF2α blocks MITF translation possibly through the inhibition of eIF2B by eIF2α.
MITF can also be directly translationally modified by the RNA helicase DDX3X. The 5' UTR of MITF contains important regulatory elements (IRES) that is recognized, bound and activated by DDX3X. Although, the 5' UTR of MITF only consists of a nucleotide stretch of 123-nt, this region is predicted to fold into energetically favorable RNA secondary structures including multibranched loops and asymmetric bulges that is characteristics of IRES elements. Activation of this cis-regulatory sequences by DDX3X promotes MITF expression in melanoma cells.
- GRCh38: Ensembl release 89: ENSG00000187098 - Ensembl, May 2017
- GRCm38: Ensembl release 89: ENSMUSG00000035158 - Ensembl, May 2017
- "Human PubMed Reference:".
- "Mouse PubMed Reference:".
- Hershey CL, Fisher DE (2004). "MITF and TFE3: members of a b-HLH-ZIP transcription factor family essential for osteoclast development and function". Bone. 34 (4): 689–96. doi:10.1016/j.bone.2003.08.014. PMID 15050900.
- Garraway LA, Sellers WR (2006). "Lineage dependency and lineage-survival oncogenes in human cancer". Nat. Rev. Cancer. 6 (8): 593–602. doi:10.1038/nrc1947. PMID 16862190.
- Hughes MJ, Lingrel JB, Krakowsky JM, Anderson KP (1993). "A helix-loop-helix transcription factor-like gene is located at the mi locus". J. Biol. Chem. 268 (28): 20687–90. PMID 8407885.
- Cheli Y, Ohanna M, Ballotti R, Bertolotto C (2010). "Fifteen-year quest for microphthalmia-associated transcription factor target genes". Pigment Cell Melanoma Res. 23 (1): 27–40. doi:10.1111/j.1755-148X.2009.00653.x. PMID 19995375.
- Moore KJ (1995). "Insight into the microphthalmia gene". Trends Genet. 11 (11): 442–8. doi:10.1016/s0168-9525(00)89143-x. PMID 8578601.
- "Genetics Home Reference". National Institutes of Health. Missing or empty
- Lister JA, Robertson CP, Lepage T, Johnson SL, Raible DW (1999). "nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate". Development. 126 (17): 3757–67. PMID 10433906.
- Delfgaauw J, Duschl J, Wellbrock C, Froschauer C, Schartl M, Altschmied J (2003). "MITF-M plays an essential role in transcriptional activation and signal transduction in Xiphophorus melanoma". Gene. 320: 117–26. doi:10.1016/s0378-1119(03)00817-5. PMID 14597395.
- Kumar S, Rao K (2012). "Waardenburg syndrome: A rare genetic disorder, a report of two cases". Indian J Hum Genet. 18 (2): 254–5. doi:10.4103/0971-6866.100804. PMC 3491306. PMID 23162308.
- Vachtenheim J, Ondrusova L (2013). "A Critical Transcription Factor in Melanoma Transcriptional Regulatory Network". Recent Advances in the Biology, Therapy and Management of Melanoma. 4: 71–73. doi:10.5772/55191.
- Smith SD, Kelley PM, Kenyon JB, Hoover D (2000). "Tietz syndrome (hypopigmentation/deafness) caused by mutation of MITF". J. Med. Genet. 37 (6): 446–8. doi:10.1136/jmg.37.6.446. PMC 1734605. PMID 10851256.
- "Melanoma Skin Cancer. " American Cancer Society, 29. Oct. 2014. Web. 15 October 2014. <http://www.cancer.org/acs/groups/cid/documents/webcontent/003120-pdf.pdf>
- Luchin A, Purdom G, Murphy K, Clark MY, Angel N, Cassady AI, Hume DA, Ostrowski MC (2000). "The microphthalmia transcription factor reulates expression of the tartrate-resistant acid phosphatase gene during terminal differentiation of osteoclasts". J. Bone Miner. Res. 15 (3): 451–460. doi:10.1359/jbmr.2000.15.3.451. PMID 10750559.
- Hoek KS, Schlegel NC, Eichhoff OM, Widmer DS, Praetorius C, Einarsson SO, Valgeirsdottir S, Bergsteinsdottir K, Schepsky A, Dummer R, Steingrimsson E (2008). "Novel MITF targets identified using a two-step DNA microarray strategy". Pigment Cell Melanoma Res. 21 (6): 665–76. doi:10.1111/j.1755-148X.2008.00505.x. PMID 19067971.
- McGill GG, Horstmann M, Widlund HR, Du J, Motyckova G, Nishimura EK, Lin YL, Ramaswamy S, Avery W, Ding HF, Jordan SA, Jackson IJ, Korsmeyer SJ, Golub TR, Fisher DE (2002). "BCL2 regulation by the melanocyte master regulator MITF modulates lineage survival and melanoma cell viability". Cell. 109 (6): 707–18. doi:10.1016/S0092-8674(02)00762-6. PMID 12086670.
- Esumi N, Kachi S, Campochiaro PA, Zack DJ (2007). "VMD2 promoter requires two proximal E-box sites for its activity in vivo and is regulated by the MITF-TFE family". J. Biol. Chem. 282 (3): 1838–50. doi:10.1074/jbc.M609517200. PMID 17085443.
- Dynek JN, Chan SM, Liu J, Zha J, Fairbrother WJ, Vucic D (2008). "Microphthalmia-associated transcription factor is a critical transcriptional regulator of melanoma inhibitor of apoptosis in melanomas". Cancer Res. 68 (9): 3124–32. doi:10.1158/0008-5472.CAN-07-6622. PMID 18451137.
- Du J, Widlund HR, Horstmann MA, Ramaswamy S, Ross K, Huber WE, Nishimura EK, Golub TR, Fisher DE (2004). "Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF". Cancer Cell. 6 (6): 565–76. doi:10.1016/j.ccr.2004.10.014. PMID 15607961.
- Meadows NA, Sharma SM, Faulkner GJ, Ostrowski MC, Hume DA, Cassady AI (2007). "The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor". J. Biol. Chem. 282 (3): 1891–904. doi:10.1074/jbc.M608572200. PMID 17105730.
- Yasumoto K, Takeda K, Saito H, Watanabe K, Takahashi K, Shibahara S (2002). "Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling". EMBO J. 21 (11): 2703–14. doi:10.1093/emboj/21.11.2703. PMC 126018. PMID 12032083.
- Sato-Jin K, Nishimura EK, Akasaka E, Huber W, Nakano H, Miller A, Du J, Wu M, Hanada K, Sawamura D, Fisher DE, Imokawa G (2008). "Epistatic connections between microphthalmia-associated transcription factor and endothelin signaling in Waardenburg syndrome and other pigmentary disorders". FASEB J. 22 (4): 1155–68. doi:10.1096/fj.07-9080com. PMID 18039926.
- Loftus SK, Antonellis A, Matera I, Renaud G, Baxter LL, Reid D, Wolfsberg TG, Chen Y, Wang C, Prasad MK, Bessling SL, McCallion AS, Green ED, Bennett DC, Pavan WJ (2009). "Gpnmb is a Melanoblast-Expressed, MITF-Dependent Gene". Pigment Cell Melanoma Res. 22 (1): 99–110. doi:10.1111/j.1755-148X.2008.00518.x. PMC 2714741. PMID 18983539.
- Vetrini F, Auricchio A, Du J, Angeletti B, Fisher DE, Ballabio A, Marigo V (2004). "The microphthalmia transcription factor (MITF) controls expression of the ocular albinism type 1 gene: link between melanin synthesis and melanosome biogenesis". Mol. Cell. Biol. 24 (15): 6550–9. doi:10.1128/MCB.24.15.6550-6559.2004. PMC 444869. PMID 15254223.
- Aoki H, Moro O (2002). "Involvement of microphthalmia-associated transcription factor (MITF) in expression of human melanocortin-1 receptor (MC1R)". Life Sci. 71 (18): 2171–9. doi:10.1016/S0024-3205(02)01996-3. PMID 12204775.
- Du J, Miller AJ, Widlund HR, Horstmann MA, Ramaswamy S, Fisher DE (2003). "MLANA/MART1 and SILV/PMEL17/GP100 are transcriptionally regulated by MITF in melanocytes and melanoma". Am. J. Pathol. 163 (1): 333–43. doi:10.1016/S0002-9440(10)63657-7. PMC 1868174. PMID 12819038.
- Chiaverini C, Beuret L, Flori E, Busca R, Abbe P, Bille K, Bahadoran P, Ortonne JP, Bertolotto C, Ballotti R (2008). "Microphthalmia-associated transcription factor regulates RAB27A gene expression and controls melanosome transport". J. Biol. Chem. 283 (18): 12635–42. doi:10.1074/jbc.M800130200. PMID 18281284.
- Du J, Fisher DE (2002). "Identification of Aim-1 as the underwhite mouse mutant and its transcriptional regulation by MITF". J. Biol. Chem. 277 (1): 402–6. doi:10.1074/jbc.M110229200. PMID 11700328.
- Carreira S, Liu B, Goding CR (2000). "The gene encoding the T-box factor Tbx2 is a target for the microphthalmia-associated transcription factor in melanocytes". J. Biol. Chem. 275 (29): 21920–7. doi:10.1074/jbc.M000035200. PMID 10770922.
- Miller AJ, Du J, Rowan S, Hershey CL, Widlund HR, Fisher DE (2004). "Transcriptional regulation of the melanoma prognostic marker melastatin (TRPM1) by MITF in melanocytes and melanoma". Cancer Res. 64 (2): 509–16. doi:10.1158/0008-5472.CAN-03-2440. PMID 14744763.
- Hou L, Panthier JJ, Arnheiter H (2000). "Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF". Development. 127 (24): 5379–89. PMID 11076759.
- Fang D, Tsuji Y, Setaluri V (2002). "Selective down-regulation of tyrosinase family gene TYRP1 by inhibition of the activity of melanocyte transcription factor, MITF". Nucleic Acids Res. 30 (14): 3096–106. doi:10.1093/nar/gkf424. PMC 135745. PMID 12136092.
- Yannay-Cohen N, Carmi-Levy I, Kay G, Yang CM, Han JM, Kemeny DM, Kim S, Nechushtan H, Razin E (June 2009). "LysRS serves as a key signaling molecule in the immune response by regulating gene expression". Mol Cell. 34 (5): 603–11. doi:10.1016/j.molcel.2009.05.019. PMID 19524539.
- Lee YN, Nechushtan H, Figov N, Razin E (February 2004). "The function of lysyl-tRNA synthetase and Ap4A as signaling regulators of MITF activity in FcepsilonRI-activated mast cells". Immunity. 20 (2): 145–51. doi:10.1016/S1074-7613(04)00020-2. PMID 14975237.
- Tshori S, Gilon D, Beeri R, Nechushtan H, Kaluzhny D, Pikarsky E, Razin E (October 2006). "Transcription factor MITF regulates cardiac growth and hypertrophy". J. Clin. Invest. 116 (10): 2673–81. doi:10.1172/JCI27643. PMC 1570375. PMID 16998588.
- Carmi-Levy I, Yannay-Cohen N, Kay G, Razin E, Nechushtan H (September 2008). "Diadenosine tetraphosphate hydrolase is part of the transcriptional regulation network in immunologically activated mast cells". Mol. Cell. Biol. 28 (18): 5777–84. doi:10.1128/MCB.00106-08. PMC 2546939. PMID 18644867.
- Hemesath TJ, Price ER, Takemoto C, Badalian T, Fisher DE (January 1998). "MAP kinase links the transcription factor Microphthalmia to c-Kit signalling in melanocytes". Nature. 391 (6664): 298–301. doi:10.1038/34681. PMID 9440696.
- Wu M, Hemesath TJ, Takemoto CM, Horstmann MA, Wells AG, Price ER, Fisher DZ, Fisher DE (February 2000). "c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi". Genes & Development. 14 (3): 301–12. PMC 316361. PMID 10673502.
- Phung B, Kazi JU, Lundby A, Bergsteinsdottir K, Sun J, Goding CR, Jönsson G, Olsen JV, Steingrímsson E, Rönnstrand L (September 2017). "D816V Induces SRC-Mediated Tyrosine Phosphorylation of MITF and Altered Transcription Program in Melanoma". Molecular Cancer Research. 15 (9): 1265–1274. doi:10.1158/1541-7786.MCR-17-0149. PMID 28584020.
- Phung, Bengt; Sun, Jianmin; Schepsky, Alexander; Steingrimsson, Eirikur; Rönnstrand, Lars (24 August 2011). Capogrossi, Maurizio C. (ed.). "C-KIT Signaling Depends on Microphthalmia-Associated Transcription Factor for Effects on Cell Proliferation". PLoS ONE. 6 (8): e24064. doi:10.1371/journal.pone.0024064. ISSN 1932-6203. PMC 3161112. PMID 21887372.
- Ngeow KC, Friedrichsen HJ, Li L, Zeng Z, Andrews S, Volpon L, Brunsdon H, Berridge G, Picaud S, Fischer R, Lisle R, Knapp S, Filippakopoulos P, Knowles H, Steingrímsson E, Borden KL, Patton EE, Goding CR (September 2018). "BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export". Proceedings of the National Academy of Sciences of the United States of America. 115 (37): E8668–E8677. doi:10.1073/pnas.1810498115. PMC 6140509. PMID 30150413.
- Morii E, Oboki K, Kataoka TR, Igarashi K, Kitamura Y (March 2002). "Interaction and cooperation of mi transcription factor (MITF) and myc-associated zinc-finger protein-related factor (MAZR) for transcription of mouse mast cell protease 6 gene". J. Biol. Chem. 277 (10): 8566–71. doi:10.1074/jbc.M110392200. PMID 11751862.
- Levy C, Nechushtan H, Razin E (January 2002). "A new role for the STAT3 inhibitor, PIAS3: a repressor of microphthalmia transcription factor". J. Biol. Chem. 277 (3): 1962–6. doi:10.1074/jbc.M109236200. PMID 11709556.
- Steingrimsson E, Tessarollo L, Pathak B, Hou L, Arnheiter H, Copeland NG, Jenkins NA (April 2002). "Mitf and Tfe3, two members of the Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development". Proc. Natl. Acad. Sci. U.S.A. 99 (7): 4477–82. doi:10.1073/pnas.072071099. PMC 123673. PMID 11930005.
- Mansky KC, Sulzbacher S, Purdom G, Nelsen L, Hume DA, Rehli M, Ostrowski MC (February 2002). "The microphthalmia transcription factor and the related helix-loop-helix zipper factors TFE-3 and TFE-C collaborate to activate the tartrate-resistant acid phosphatase promoter". J. Leukoc. Biol. 71 (2): 304–10. PMID 11818452.
- Xu W, Gong L, Haddad MM, Bischof O, Campisi J, Yeh ET, Medrano EE (March 2000). "Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9". Exp. Cell Res. 255 (2): 135–43. doi:10.1006/excr.2000.4803. PMID 10694430.
- Razin E, Zhang ZC, Nechushtan H, Frenkel S, Lee YN, Arudchandran R, Rivera J (November 1999). "Suppression of microphthalmia transcriptional activity by its association with protein kinase C-interacting protein 1 in mast cells". J. Biol. Chem. 274 (48): 34272–6. doi:10.1074/jbc.274.48.34272. PMID 10567402.
- Falletta, Paola; Sanchez-del-Campo, Luis; Chauhan, Jagat; Effern, Maike; Kenyon, Amy; Kershaw, Christopher J.; Siddaway, Robert; Lisle, Richard; Freter, Rasmus (1 January 2017). "Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma". Genes & Development. 31 (1): 18–33. doi:10.1101/gad.290940.116. ISSN 0890-9369. PMC 5287109. PMID 28096186.
- Phung, Bengt; Cieśla, Maciej; Sanna, Adriana; Guzzi, Nicola; Beneventi, Giulia; Cao Thi Ngoc, Phuong; Lauss, Martin; Cabrita, Rita; Cordero, Eugenia (June 2019). "The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma". Cell Reports. 27 (12): 3573–3586.e7. doi:10.1016/j.celrep.2019.05.069. PMID 31216476.