TUBA4A gene analysis in sporadic amyotrophic lateral sclerosis: identification of novel mutations

Dear Sirs,

Amyotrophic lateral sclerosis (ALS) is a late-onset disease caused by motor neuron degeneration with no effective therapies [1]. Approximately, 5–10 % of cases are familial (FALS), whereas the majority of patients are sporadic (SALS). ALS exhibits an extreme genetic heterogeneity and at least 25 genes are associated to familial forms, with C9orf72 and SOD1 representing the most common mutated genes [2].

Recently, by performing an exome-wide, case-control burden analysis of rare variant in FALS index cases, we identified an excess of patient variants (7/635) in TUBA4A gene, encoding for a member of the alpha-tubulin family [3]. Functional studies showed that TUBA4A mutations exert deleterious effects on microtubule network and dynamics in primary motor neurons. By extending TUBA4A genetic analysis to 1355 sporadic cases of different origin, we identified one additional variant (p.Gly43Val) with a mild effect on microtubule cytoskeleton in an Italian patient [3].

These data led us to further assess the involvement of TUBA4A gene in sporadic cases by analyzing a large cohort of 1106 SALS of Italian origin, including 43 patients with concomitant fronto-temporal dementia (ALS-FTD).

Our mutational screening revealed the presence of four novel heterozygous variants in four patients (Table 1). Three were missense mutations (p.Val7Ile; p.Thr349Ser and p.Asp438Asn) which determined amino-acid substitutions at evolutionarily conserved residues (“Online Resource”), while the fourth variant (c.226+4A>G) was a donor splice site mutation in in-tron 2. These variants were absent in 3960 in-house Italian controls as well as in 7595 individuals from public databases (1000 Genome project and NHLBI GO Exome Sequencing Project). Other synonymous variants and previously reported polymorphisms were also detected (“Online Resource”).

Table 1.

TUBA4A variants identified in SALS Italian patients

Sample ID	Sex	Age at onset	Disease duration	Site of onset	Cognitive impairment	Variant	SIFT prediction	Mutation taster	PolyPhen2 prediction	BDGP Wild type/mutant	ASSP wild type/mutant
B1103	M	57	14 years	Spinal	No	IVS2+4A>G c.226+4A>G	n/a	n/a	n/a	0.76/-	10.264/7.612
P937	F	n/a	n/a	n/a	n/a	p.Val7Ile c.19C>A	Tolerated	Disease causing	Benign	n/a	n/a
N5214	F	62	12 months	Bulbar	No	p.Thr349Ser c.l045A>T	Damaging	Disease causing	Possibly damaging	n/a	n/a
N6287	M	59	26 months	Bulbar	Yes	p.Asp438Asn c.1312G>A	Damaging	Disease causing	Benign	n/a	n/a

BDGP Berkeley Drosophila Genome Project: Splice Site Prediction by Neural Network, ASSP Alternative Splice Site Predictor, n/a not available, - the constitutive splice site is not recognized in mutant sequence

In silico analysis predicted a possibly damaging effect of three novel missense mutations on TUBA4A protein and of the c.226+4A>G variant on exon 2 splicing (Table 1). We proved that this variant abolished the original donor splice site resulting in exon 2 skipping using a minigene splicing assay (Fig. 1).

Fig. 1.

Minigene splicing assay for TUBA4A intronic variant c.226+4A>G. a Schematic representation of the pTB minigene system, in which light blue boxes represent a-globin exons and thick lines are introns. TUBA4A exon 2 (orange box) along with part of flanking introns was subcloned into the NdeI restriction site of the pTB vector. The primers a 2–3 and Bra2rev, used for RT-PCR analysis, are indicated as thin blue arrows in the pTB map. b HEK293 cells were transfected with wild-type (WT) or mutant (MUT) TUBA4A hybrid minigene, or with the empty vector (pTB), as indicated. The size of the transcripts, with TUBA4A exon 2 inclusion or exclusion, is indicated on the right side of the gel. The molecular weight marker (100pb DNA ladder, Life Technologies) is reported

All patients carrying TUBA4A mutations had a classical ALS phenotype, with upper and lower motor neuron signs (full clinical information is listed in “Online Resource”). Notably, one case also showed mild cognitive impairment, adding further evidence that TUBA4A mutations in ALS may be associated to the ALS-FTD continuum, as previously observed [3].

In conclusions, here we report the identification of novel TUBA4A variants with predicted deleterious effects on protein function in a series of SALS Italian patients. Although functional studies are needed to determine their pathological effect on microtubule network, our results further support the role of TUBA4A gene in ALS. Together with PFN1 gene [4], mutations in TUBA4A indicate that defects in neuronal cytoskeleton architecture represent one of the pathogenic mechanisms triggering neurodegeneration.