Angiogenin Loss-of-Function Mutations in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that typically presents in the fifth to sixth decades of life with upper and lower motor neuron signs. Initially, there are symptoms that include distal muscle weakness and wasting, increased muscle tone with hyperreflexia, and at times diaphragmatic and/or bulbar weakness. Atypical forms can include symptoms of dementia, parkinsonism, or both. All forms of ALS inexorably progress to generalized amyotrophy, culminating in respiratory failure and death after an average duration of 3 to 5 years.1 The incidence of ALS is estimated at 0.4 to 1.8 per 100,000 people.2,3 Approximately 80 to 90% of ALS cases occur in individuals with no known family history, whereas the remaining cases are attributable to familial inheritance in either an autosomal dominant or recessive manner.3,4 Mutations in the Cu/Zn superoxide dismutase gene 1 (ALS1; SOD1; OMIM 147450), have been identified in 12 to 23% of familial5–7 and in 0 to 7% of sporadic8–10 ALS patients. Currently, SOD1 is the only known autosomal dominant gene in which mutations have been functionally associated with ALS, although three other loci have been identified for typical autosomal dominant ALS by linkage (ALS3, 18q21, OMIM 606640; ALS6, 16q12, OMIM 608030; and ALS7, 20ptel-p13, OMIM 608031).2,3 Other dominantly inherited genetic loci, associated with an atypical ALS phenotype, have also been identified (ALS with dementia/parkinsonism, MAPT, OMIM 157140; progressive lower motor neuron disease, DCTN1, OMIM 601143; and ALS8, VAPB, OMIM 608627). In autosomal dominant ALS with frontotemporal dementia (OMIM 105550), genetic linkage has been reported to 9q21-q22.11 Mutations in the SETX gene (OMIM 608465) have been identified in juvenile-onset autosomal dominant ALS. Lastly, genetic loci identified for juvenile-onset autosomal recessive disease include ALS2 (ALS2, 2q33, OMIM 606352) and linkage for ALS5 to 15q15.1-q21.2,3 Recent linkage analysis in Irish and Scottish ALS populations identified chromosome 14q11.2 as a candidate region and then angiogenin (ANG), a 14.1kDa angiogenic ribonuclease (RNase), as an ALS candidate gene.12,13 Seven heterozygous missense mutations in ANG were identified by sequence screening of 1,629 patients with ALS.13 Analysis of the ANG crystal structure suggested that these mutations may disrupt the structure and result in functional loss. However, the functional consequences of these mutations are unknown.13 We now report herein the identification of four mutations in the coding region of the ANG gene on screening an independent cohort of 298 SOD1-negative ALS patients. Three of these mutations occur in the mature protein and one in the signal peptide sequence. Using angiogenesis, ribonucleolysis, and nuclear translocation assays, we demonstrate that these mutations result in complete loss of function. Moreover, we show ANG expression in both endothelial cells and motor neurons of normal human fetal and adult spinal cord. Our data suggest that ANG plays a role in motor neuron health and provide evidence that ANG mutations, identified in ALS patients, are associated with functional loss of angiogenic activity.