Chapter # EVOLUTION AND ORIGIN OF NEUROFIBROMIN, THE PRODUCT OF THE NEUROFIBROMATOSIS TYPE 1 (NF1) TUMOR-SUPRESSOR GENE

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1 142 Part 5 Chapter # EVOLUTION AND ORIGIN OF NEUROFIBROMIN, THE PRODUCT OF THE NEUROFIBROMATOSIS TYPE 1 (NF1) TUMOR-SUPRESSOR GENE Golovnina K. *1, Blinov A. 1, Chang L.-S. 2 1 Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia; 2 Center for Childhood Cancer, Children s Research Institute, Children s Hospital and Department of Pediatrics, The Ohio State University, USA * Corresponding author: ksu@bionet.nsc.ru Key words: neurofibromin, the Neurofibromatosis type 1 (NF1) gene, RasGAP protein family, GTPase-activator proteins for Ras-like GTPase, phylogeny, BLAST SUMMARY Motivation: Neurofibromatosis type 1 (NF1) is a common genetic disorder, which predisposes affected individuals to a variety of clinical features including tumors of the central and peripheral nervous systems. The product of the NF1 gene, neurofibromin, is a tumor suppressor which most likely acts through the interaction of its GTPase activating protein (GAP) related domain (GRD) with RAS to regulate cellular growth. While clinical features of NF1 as well as functional activity of human neurofibromin are intensively studied now little is understood about its evolution, diversity, and overall distribution among different taxa. Results: By combining bioinformatic and phylogenetic approaches, we demonstrated that NF1 homologs are present across a wide range of eukaryotic lineages. We observed 26 similar to NF1 amino-acid sequences from Chordata, Echinodermata, Arthropoda, Platyhelmintes and Fungi taxons. Taking into account a presence of NF1 gene in fungi, we can suggest the derivation of this gene before the Metazoan origin. In this case, an absence of NF1 in Nematoda and Mollusca should be a result of this gene elimination. INTRODUCTION Clinical features of NF1. Neurofibromatosis type I (NF1) or von Recklinghausen neurofibromatosis is the most common cancer predisposition syndrome affecting the nervous system with the incidence of 1 in 3000 worldwide (Gutmann, 2001). Typical manifestations include café au lait spots (hyperpigmented macules), cutaneous and subcutaneous neurofibromas (benign tumors), and malignancies of the central and peripheral nervous systems. The less common abnormalities observed in NF1 patients include learning disabilities (although frank mental retardation is rare) and skeletal abnormalities such as scoliosisand pseudoarthrosis (Skuse, Cappione, 1997). In addition to these features, children with NF1 can present within the first 6 years of life with lowgrade glial tumors involving the optic parthway. Two intriguing features of NF1 are the wide range of potentially affected tissues and the great variation in expressivity of disease traits across those affected. To date, the underlying source of this variation remains somewhat unclear, but evidence suggests that aberrations in normal NF1 RNA processing may be involved (Skuse, Cappione, 1997).

2 Comparative and evolutionary genomics and proteomics 143 Genetic features of NF1. Early insights into the patogenesis of NF1-associated tumors began with the identification of the NF1 gene in 1990 (Wallace et al., 1990). The NF1 gene itself, located at 17q11.2, encompasses >300 kb of human chromosome 17 (Li et al., 1995). The 60 exons which constitute the human NF1 gene give rise to several alternatively spliced transcripts. Within the central portion of the NF1 encoded protein, neurofibromin, lies a region with homology with the mammalian GTPase activating proteins (GAP) and the yeast inhibitor of RAS proteins 1 and 2 termed the GAP related domain (GRD). Neurofibromatoses. Discussions about neurofibromatoses include two the most common forms of this disorder: neurofibromatosis 1(NF1) and neurofibromatosis 2 (NF2). Individuals with two inherited cancer syndromes, NF1 and NF2 develop both benign and malignant tumors. The corresponding genes mutated in these two disorders encode tumor suppressor proteins, termed neurofibromin (NF1) and merlin (NF2), which have a similar function to regulate cell growth and differentiation. While both genes are not related and located on the different chromosomes in human, nothing is known about their relationships, distribution and function in other organism as well as about their origin. In the previous study we investigated evolution of merlin and postulated its origin in early metazoan (Golovnina et al., 2005). Here we represent the initial phylogenetic research of NF1 protein and its homologs based on bioinformatics approaches. METHODS AND ALGORITHMS BLAST search. Initial sequences of genes and proteins of interest from various organisms were identified by performing multiple TBLASTN and BLASTP (Altschul et al., 1997) searches against GenBank ( Ensembl ( ) and wormbase ( databases. In each case putative H.sapiens NF1 protein were used as the query sequence. Only NF1-like representatives of insects were located by D. melanogaster NF1 protein homology. To obtain more information we then searched the desirable sequences across genomic databases of completely or partially sequenced genomes available at The Sanger Institute ( and The Institute for Genomic Research (TIGR) ( Doe Joint Genome Institute (JGI) ( The Broad Institute ( The predicted nucleotide and aminoacid sequences of many species were assembled manually using available contigs and assemblies of genomes by homology to query sequence. Alignments and phylogeny. The Clustal X program (Thompson et al., 1997) was used to align the characterized or predicted protein sequences from different species. All alignments were corrected for obvious alignment ambiguity The resultant alignment contained 3888 aligned positions and was used to construct phylogenetic tree. Phylogenetic analysis was carried out using the Neighbor-Joining method in MEGA 3.1 program (Kumar et al., 2004). RESULTS AND DISCUSSION In total, 22 NF1-like sequences, that have overall homology, from Deuterostomia, Arthropoda, Platyhelmintes have been located in the present investigation. The main criterion for identification these sequences was their evident homology to known NF1 proteins. Moreover, we have found four Fungi proteins that show not so strongly similarity to NF1-like sequences. Five of 22 NF1-like proteins are experimentally annotated, namely H. sapiens NF1, M. musculus NF1, R. norvegicus NF1, T. rubripes NF1, and D. melanogaster NF1. Eight homologs were bioinformatics predicted previously and were located by initial BLASTP search across NCBI database

3 144 Part 5 (P. troglodytes, C. familiaris, G. gallus, A. gambiae), as well as NF1-related protein of N. crassa and three other RasGAPs of fungi. The rest sequences were assembled manually by parts using available paired scaffolds, contigs, and assemblies of the sequenced genomes together with protein and EST databases. Based on the obtained NF1 sequences and the close related proteins the phylogenetic tree was constructed (Fig. 1). RasGap family members from another groups were included in our analysis as an outgroup to show a homology of all obtained NF1- like sequences. We conducted phylogenetic analysis including only RasGap domain and corresponding sequences of newly identified proteins in view of possible different origin of some other domains except RasGap (Fig. 2) Both trees are similar in topology with small alterations in Fungi group. Figure 1. Phylogenetic analysis of NF1 homologs and its close related proteins from RasGAP group. NF1 clade is shown by solid line on the right. The dashed line denotes observed fungi proteins that are similar to NF1. UniProt accession numbers for full length sequences are represented in bold letters, GenBank in regular.

4 Comparative and evolutionary genomics and proteomics 145 Figure 2. Phylogenetic analysis of NF1 homologs and its close related proteins from RasGAP group based on RasGap domain sequences. Fungi proteins that have similarity with NF1-like sequences are marked with dark round. Phylogenetic analysis demonstrate a distribution of NF1 homologs across all investigated and available to date the major metazoan (chordata, urochordata, echinodermata, insects, and platyhelmintes) and fungi lineages. Among all species investigated, no NF1 sequences have been found in round worms (C. elegans, C. briggsae, C. remanie, B. malayi) and mollusk (B. glabrata). There are two possible explanations: (i) the derivation of NF1 gene occured after a separation of both Nematoda and mollusca or (ii) it was lost specifically in these groups. In C. elegans genome were previously annotated RasGAP family members (GAP1 and GAP2) that have homologs in C. briggsae genome. They are clasterized together with synras group of proteins that means their evolutionary relatedness. The presence of NF1-related sequences in fungi is disputable. NF1-like proteins have been found in four species of fungi kingdom, belonging to both Ascomycota and Basidiomycota. These amino-acid sequences have overall similarity to other NF1-like proteins but not so strong besides sequences corresponding to RasGap domain are more similar to other fungi RasGap proteins (Fig. 2). One of them (N. crassa) was previously annotated as NF1-related protein. On the other hands, no NF1-like sequences were detected in the rest fungi species examined.

5 146 Part 5 Therefore, the root of neurofibromin origin is unclear now and with the great progress in genome sequencing projects it promised to be more definite. Previously, no merlin homologs (another tumor suppressor) in fungi were observed. ACKNOWLEDGEMENTS This study was supported by grants from the US Department of Defense Neurofibromatosis Research Program. REFERENCES Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res., 25, Golovnina K., Blinov A., Akhmametyeva E.M., Omelyanchuk L.V., Chang L.-S. (2005) Evolution and origin of merlin, the product of the neurofibromatosis type 2 (NF2) tumor-suppressor gene. BMC Evolutionary Biology, 5, 69. Gutmann D.H. (2001) The neurofibromatosis: when less is more. Hum. Mol. Genet., 10, Kumar S., Tamura K., Nei M. (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics, 5, Li Y., O Connell P., Breidenbach H.H., Cawthon R., Stevens J., Xu G., Neil S., Robertson M., White R., Viskochil D. (1995) Genomic organization of the neurofibromatosis 1 gene (NF1). Genomics, 25, Skuse G.R., Cappione A.J. (1997) RNA processing and clinical variability in neurofibromatosis type I (NF1). Hum. Mol. Genet., 6, Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res., 15, Wallace M.R., Marchuk D.A., Andersen L.B., Letcher R., Odeh H.M., Saulino A.M., Fountain J.W., Brereton A., Nicholson J., Mitchell A.L. et al. (1990) Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science, 249,