POSTER NO: 420

Identification and characterization of the ALS2 causative gene that encodes a protein containing multiple GEF domains

¹S. Hadano, ²C.K. Hand, ¹H. Osuga, ¹Y. Yanagisawa, ¹A. Otomo, ³R.S. Devon, ¹N. Miyamoto, ¹J. Shouguchi-Miyata, ¹Y. Okada, ³R. Singaraja, ⁴D.A. Figlewicz, ⁵T. Kwiathowski, ⁵B.A. Hosler, ⁶T. Sagie, ⁷J. Skaug, ⁸J. Nasir, ⁵R.H. Brown Jr., ⁷S.W. Scherer, ²G.A. Rouleau, ³M.R. Hayden, ¹J.-E. Ikeda
¹SORST, Japan Science and Technology Corporation, and Department of Molecular Neuroscience, The Institute of Medical Sciences, Tokai University, Kanagawa, Japan, ²Centre for Research in Neuroscience, McGill University, Canada, ³Department of Medical Genetics, and Centre for Molecular Medicine and Therapeutics, University of British Columbia, Canada, ⁴Departments of Neurology and Neurobiology and Anatomy, University of Rochester Medical Center, U.S.A., ⁵Day Neuromuscular Research Laboratory, MGH-EAST, U.S.A, ⁶Pediatric Neurology and Metabolic Neurogenetic Clinic, Israel Sackler School of Medicine, Tel Aviv University, Israel, ⁷Department of Genetics, The Hospital for Sick Children, Canada, ⁸Division of Genomic Medicine, University of Sheffield, U.K.

Amyotrophic lateral sclerosis 2 (ALS2) was originally reported in a large consanguineous Tunisian kindred, and was characterized by a loss of upper motor neurons and spasticity of limb and facial muscles accompanying distal amyotrophy of hands and feet. To understand the molecular mechanism for the pathogenesis of ALS2 and other forms of ALS, identification of the gene and mutation that links to ALS2 is essential. In this study, we report the identification of two independent deletion mutations in the coding exons for the new gene, ALS2, in the Tunisian and Kuwaiti ALS2 families.

We have identified 395 exons from 42 non-overlapping transcripts within 3 Mbp of the ALS2 candidate interval between D2S116 and D2S2237 on human chromosome 2q33. A single nucleotide deletion (138delA) linked to the Tunisian ALS2 was identified in the coding exon of the novel gene, ALS2. This deletion clearly co-segregated with the ALS2 phenotype and was not seen in 533 normal individuals. Further, a second deletion mutation (1425_1426delAG) was found in the Kuwaiti ALS2/JPLS family. Both deletion mutations disrupt the reading frames in the ALS2 transcript, suggesting mutations cause deleterious effect on the encoded protein (a loss-of-function mutation).

ALS2 comprises 33 introns and 34 exons and resides within 80.3kb of genomic DNA. Sequence of the ALS2 transcript encompassed 6,394 nucleotides with a single open reading frame (ORF: 4,974 nucleotides long, 124 - 5,097 nt), and is predicted to encode a 184 kD protein consisting of 1,657 amino acids. We also identified a shorter transcript for ALS2 that encompassed 2,651 nucleotides. Consistent with these results, we identified, by Northern blot analysis, two transcripts of approximately 6.5kb and 2.6kb in various adult human tissues. Both transcripts showed a similar pattern of expression with the large 6.5kb transcript showing highest expression in the cerebellum. In addition to these two forms, five novel splicing variants were also identified. The ALS2 gene is predicted to encode a putative small GTPase regulator containing three independent domains for the guanine nucleotide exchange factors (RanGEF, RhoGEF, and Rab5GEF). Thus, it is tempting to speculate that the ALS2 protein acts as a regulator/activator of particular small GTPases and modulates the microtuble assembly, membrane organization and/or trafficking in cells including neurons. Identification of the protein function will offer clues to understand a molecular mechanism underlying the pathogenesis of ALS2.