A novel Angiogenin gene mutation in a sporadic patient with amyotrophic lateral sclerosis from southern Italy

1. Introduction 

Recently, Greenway and colleagues [1] reported an allelic association and different novel mutations in the Angiogenin gene (ANG) suggesting a role for Angiogenin in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). ANG gene maps to chromosome 14q11.2 and consists of only one exon with a translation length of 147 residues. The protein, originally identified as a molecule involved in the formation of blood vessels, now is demonstrated to have important neuroprotective activities [2].

2. Patients and methods 

We analyzed the ANG gene in a clinical ALS sample (SOD1 mutation-negative) from southern Italy and in ethnic- and sex-matched controls. One hundred and sixty-three patients with ALS (8 familial ALS and 155 sporadic cases, 84 men and 79 woman; mean age 54.52years, SD 12.14), diagnosed according to the El Escorial criteria, and 332 healthy controls from the same geographical area (195 men and 137 woman; mean age 49.75years, SD 20.59) were analyzed. All participants provided a signed informed consent, after which a DNA extraction and genotyping was performed using standard protocols.

3. Results 

The sequence analysis of the ANG gene (GenBank genome sequence M11567 – version GI: 178249) revealed a novel mutation g.1811G→A in the first methionine of the signal peptide of ANG gene in a sporadic ALS patient (SALS) (Fig. 1). This variation was also confirmed by clonation of amplified product into pGEM-T vector with a loning Promega Kit, and subsequently sequenced. This novel mutation, possibly disrupting the signal important for protein maturation, is predictive of the aminoacid change of methionine in isoleucine (ATG→ATA) although the effect on the protein level is unknown. This mutation was not present in 332 controls or in the NCBI database. The patient is a 68-year-old man with a five-year history of weakness in upper limbs and fasciculation in both arms and shoulders. Neurological examination showed moderate atrophy, weakness and diffused fasciculation of the proximal and distal muscles of the upper limbs, while bulbar functions and muscle strength in the lower limbs were clinically normal. Tendon reflexes were brisk in the four limbs; bilateral plantar response, and sensation were normal. His family history and past medical history were negative for dementia, motor neuron diseases and other neurological disorders. His parents were not consanguineous; because both deceased and no cell line was available for testing, we cannot determine whether the mutation occurred de novo or was inherited. Two of six proband’s siblings are still alive but only one underwent the genetic analysis resulting negative for the ANG gene mutation. EMG showed active and chronic diffuse neurogenic activities in both upper and lower limbs. Motor and sensory conduction studies were within normal range.

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Fig. 1. Sequence electropherograms of the ANG gene. (a) Sequence of a normal subject; (b) sequence of the SALS patient showing the nucleotide substitution ATG→ATA. The arrow indicates the substitution site.

The ANG sequence analysis also confirmed the presence of the synonymous rs11701 SNP, a variant previously described both in ALS and controls [3]. The distribution of rs11701 SNP genotypes was in Hardy–Weinberg equilibrium in both groups and no difference was found in sex distribution. There were significant differences in allelic (G: 17.5% vs 11.7% p-value=0.013) and genotype (T/T: 66.9% vs 77.7%; T/G: 31.3% vs 21.1%; G/G: 1.8% vs 1.2%, p-value=0.035) distribution of the rs11701 SNP between patients and controls. Moreover, when ALS population was stratified in SALS and FALS, there was a significant difference in allelic and genotype distribution between FALS patients and controls (G: 43.8% vs 11.7%; p-value=0.005) whereas no difference was observed between SALS and controls (G: 16.1% vs 11.7%; p-value=0.201). Individuals with FALS carrying the G allele had a greater risk for ALS than those carrying the T allele (OR: 5.74, 95% CI: 2.07–15.95). However, we cannot exclude that the association between the G allele and FALS could be due to the mismatch in the sample size of FALS and control samples (8 vs 332).

Previously described SNPs rs17560 and rs2228653 were observed in both ALS and in control subjects (1% and 0.3%, respectively). Furthermore, we found the aminoacid substitution (I46V) in five control subjects (2%) confirming that this variation represents a polymorphism [4].

4. Discussion 

In the current study, we report a novel mutation in the ANG gene in one subject with SALS. To date, mutations that affect the signal peptide have been described and characterized in association with various human diseases [5], [6]. This mutation, that possibly affects Angiogenin function, confirms the role of the ANG gene in developing ALS, and gives the first evidence of a link between ALS and angiogenesis in the Italian population.

Furthermore, our results reveal an association between FALS and rs11701 SNP in ANG gene but, despite previous data [1], not in SALS patients. This suggests that a difference in the genetic background between different populations may be an important factor leading to different results in genetic association studies in ALS.