Missense mutation in ATXN2 gene (c.2860C > T) in an amyotrophic lateral sclerosis patient with aggressive disease phenotype

Abstract

Background

ALS symptoms have been previously described only in the context of ATXN2 CAG expansions, whereas missense mutations of the gene have never been described in ALS patients.

Case presentation

We identified a novel missense mutation (c.2860C > T) of ATXN2, for which in silico analysis showed a possible pathogenic effect on protein expression, in a patient presenting an aggressive disease phenotype.

Discussion

Our findings raise the possibility for unknown genetic factors interacting with ATXN2 mutations, or for an autonomous pathogenic role for this specific point mutation in ATXN2 gene in driving the clinical phenotype toward ALS. We also found that stress granules in the fibroblasts from the patient entrapped higher amounts of defective ribosomal products compared to fibroblasts from three healthy subjects, suggesting that ATXN2 mutation-related toxicity may have implication in protein quality control.

Appendix

Appendix. Methods for genetic screening

Genomic DNA was extracted from venous peripheral blood lymphocytes by standard procedures. Preliminary mutational screening for C9orf72 gene was performed with repeat-primed PCR (RP-PCR) analysis in order to identify pathological expansions of the GGGGCC, ruling out pathogenetic expansion. Sequencing was performed using an Illumina MiSeq sequencing device. Variants with possible pathogenetic significance were verified by Sanger method on the sequencer ABI PRISM 3130 XL. The following causative and susceptibility genes in motoneuron diseases were analyzed: AARS, ALS2, ANG, ANXA11, AP5Z1, APEX1, ARHGEF28, ARPP21, ASAH1, ATXN2, BAG3, BSCL2, CFAP410 (C21ORF2), CHCHD10, CHMP2B, CRYM, CYP27A1, CYP7B1, DAO, DCTN1, DHTKD1, DPP6, ELP3, EPHA4, ERBB4, FIG4, FUS, GARS, GRN, HNRNPA1, HNRNPA2B1, HNRNPA3, HSPB1, HSPB3, HSPB8, IGHMBP2, KIF5A, MAPT, MATR3, MFN2, NEFH, NEK1, OPTN, PFN1, PNPLA6, PRNP, PRPH, PSEN1, PSEN2, REEP1, SETX, SIGMAR1, SOD1, SPAST, SPG11, SPG21, SPG7, SPTLC1, SQSTM1, TAF15, TARDBP, TREM2, TRPV4, TUBA4A, UBQLN2, VAPB, VCP, and ZFYVE26.

Data analysis was performed using (a) MiSeq Reporter software 2.4.60 (Illumina) for quality control, alignment against hg19 reference genome, and variant calling; (b) VariantStudio software 2.2 (Illumina) for variant annotation; (c) CLC GenomicsWorkbench software (CLCbio, Qiagen), for quality control and coverage analysis.

Genetic variants with frequency > 1% in dbSNP or in 1000 Genome project or in Exome Variant Server were classified as polymorphisms, and were excluded from subsequent analyses. Variants considered as class I (benign) or II (likely benign) were not considered, even if minor allele frequency (MAF) was < 1% in the same population databases.