Novel and Recurrent Mutations in a Cohort of Chinese Patients With Young-Onset Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and spinal cord. More than 25 ALS-related genes have been identified, accounting for approximately 10% of sporadic ALS (SALS) and two-thirds of familial ALS (FALS) cases. Several recent studies showed that genetic factors might have a larger contribution to young-onset ALS than to ALS cases overall. However, the genetic profile of young-onset ALS patients is not yet fully understood. Here, we investigated a cohort of 27 young-onset ALS patients (onset age < 45 years) through whole-exome sequencing (WES). Genetic analysis identified pathogenic variants of FUS (25.9%), SOD1 (22.2%), TARDBP (3.7%), and VCP (3.7%) in 27 young-onset ALS patients. Of 12 identified types of mutations, c.1528A > C in FUS and c.266G > A in VCP were novel. All of the cases in this study reflect a monogenic origin with an autosomal dominant mode of inheritance. Notably, a novel de novo missense mutation, c.1528A > C (p.K510Q), in FUS was identified in a 29-year-old ALS patient. Expression of the K510Q mutant FUS resulted in cytoplasmic mislocalization of FUS in cultured cells and induced neural toxicity in a fly model. This study provides further evidence of the genetic profile of young-onset ALS patients from China and expands the mutational spectrum of the FUS gene, with one new K510Q mutation identified.

Keywords: Drosophila model; amyotrophic lateral sclerosis; c.1528A > C (p. K510Q); fused in sarcoma; novel mutation; young-onset.

FIGURE 1

Mutation spectrum of Chinese young-onset ALS and identification of the K510Q FUS mutation. (A) Pie chart showing that 55% of young-onset ALS patients had identified validated mutations, consisting of 22% familial ALS (FALS) and 33% sporadic ALS (SALS). (B) Percentage of each validated gene in FALS and SALS in this cohort. SOD1 has the highest mutation frequency in FALS and FUS in SALS. (C) Sequencing results of amplified genomic DNA from the patient and his parents. The novel FUS mutation c.1528A > C (p. K510Q) was identified in the patient. Mutated nucleotide was indicated by arrowhead. The reading frame depicting the corresponding amino acid substitution is shown on the top of the electropherogram. (D) The family pedigree of the patient with ALS. The proband was indicated by arrowhead. (E) Schematic diagram of the full-length human FUS protein and the K510Q and P525L mutations located in the nuclear localization signal domain (NLS).

FIGURE 2

The K510Q mutant FUS is mislocalized in cytoplasm in HEK293 and HT22 cells. (A) HEK293 cells were transfected with EGFP-tagged Wt, P525L, or K510Q FUS. P525L or K510Q mutant FUS proteins were mislocalized in the cytoplasm. (B) Mouse neuronal cells (HT22) were transfected with EGFP-tagged Wt, P525L, or K510Q FUS. Cytoplasmic inclusions of mutant FUS were found in cells expressing P525L or K510Q mutant FUS. Scale bar: 10 μm.

FIGURE 3

Over-expression of the K510Q mutant FUS in retinal neuron induced neural toxicity in a transgenic fly model. (A) Microscopic images of fly eyes expressing Ctr and K510Q mutant FUS. Fly genotypes: Ctr: GMR-Gal4/W1118; FUS^K510Q: GMR-Gal4/UAS-K510Q-FUS. (B) Western blot analysis of FUS protein in transgenic fly eyes. The pan-neuronal marker Elav was used as a loading control.