Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a neurodegenerative disorder that affects the upper and lower motor neurons in the motor cortex, brain stem, and spinal cord [20]. ALS was first described in the mid-1800s as a rapidly progressing motor neuron disease, with phenotypes of varying severity. Affected patients present muscle weakness, atrophy, and a general progressive paralysis. Eventually, most develop respiratory failure which leads to death on average 3 to 5 years after the onset of symptoms [45]. As yet, there is no effective treatment or pre-symptomatic diagnostic test for ALS [11].

Approximately 90–95% of ALS patients suffer from a sporadic form of ALS (sALS), which has both an environmental etiology and a likely strong genetic component [23]. The remaining 5–10% of ALS patients exhibit a clear genetic etiology for the disease, also known as familial ALS (fALS). Genomic variants in the SOD1 gene have been shown to lead to fALS, with an autosomal dominant mode of inheritance. To date, more than 170 different SOD1 genomic variants have been reported, affecting ~ 13% of fALS and ~ 1% of sALS patients [40]. Furthermore, variants in other genomic loci, such as FUS [27], TARDBP [25], and more recently C9orf72 [36], have also been implicated in fALS. In the case of C9orf72, a non-coding GGGGCC expansion appears to have a very strong association with both fALS and sALS, with an overall prevalence of 40 and 5–20% in fALS and sALS patients, respectively [39].

The etiology of sALS is quite complex. A variety of different factors, including age, overall lifestyle, occupation, and/or exposure to xenobiotics, appear to influence the risk of developing sALS, but no specific chemical or toxin has been identified as a direct cause of ALS [44]. In addition to environmental factors, sALS also has a substantial genetic component. Genome-wide association studies have so far identified over 100 genomic loci that could yet prove to be linked to ALS disease predisposition with low-to-medium penetrance, supporting the notion of pronounced heritability for ALS [reviewed in [28]]. From these genomic loci, SQSTM1 [19], UBQLN2 [14], VCP [12], ANG [29], PRPH [38], DCTN1, TAF15, and SETX [10] appear to be more frequently mutated in sALS than in fALS, with an overall prevalence of between 4 and < 1%; these findings have been confirmed in several studies/cohorts as well as in functional and animal studies. Furthermore, the co-occurrence of genomic variants in addition to the presence of de novo pathogenic variants in sALS brings the estimate of overall heritability for ALS to as high as 60% [3, 4]. The identification of the entire lexicon of underlying genetic defect(s) in both fALS and sALS patients and their families would greatly improve disease diagnosis and potentially disease management.

There is little knowledge of the genetic basis of ALS in fALS and sALS patients of Greek origin. Although the incidence of ALS in Greek patients is comparable to other European populations (~ 1–2 per 100,000), only a single study has so far been performed, highlighting a rather high prevalence of the C9orf72 GGGGCC repeat expansion in Greek ALS patients [35]. Here, we report a novel genomic locus, FTO, that appears to be associated with sALS. Genomic data comparison with sALS patients of Greek, Sardinian, and Turkish descent strongly suggest that this association may represent a founder effect that is specific to the Greek population.

sALS is polygenic with a complex interplay with environmental and other factors. Previous studies have proposed genetic associations of a variety of genes with the etiology of sALS. In this article, we adopted a three-step approach to delineate the association of genomic variants with sALS in patients of Greek origin.

Using a next-generation sequencing-based genomics approach, we identified 174 variants that were only present in the initial sALS patients studied. We opted to focus on the FTO rs2892469 (C>T) and rs1861869 (G>C) and TBC1D1 rs6850200 (C>A) variants, based on pathway analysis. Our replication analyses confirmed that the FTO but not the TBC1D1 genomic variants maintained their statistical significance as far as their association with sALS was concerned. Data stratification considering (i) the age of onset (below or above 50 years old), (ii) El Escorial criteria, and (iii) spinal or bulbar ALS onset did not affect our findings (not shown).

We subsequently attempted to explore whether the FTO gene variants identified as being associated with sALS in Greek patients might also be associated with sALS in patients of Turkish and Sardinian descent. However, genetic analysis of all FTO and TBC1D1 variants failed to reach statistical significance, even when stratified for SOD1, when Sardinian and Turkish sALS patients were considered. This may be due to a founder effect in the Greek population.

In order to explore the functional significance of our findings, we measured the FTO and TBC1D1 gene expression levels in several human cell types, namely fibroblasts, embryonic stem cells (ESCs), astrocytes, neural PAX6+ progenitors, stem cell-derived purified motor neurons at three different stages of development (Hb9::GFP on days 0, 16, and 30), and stem cell-derived cortical neurons (NKX21 on days 45 and 50). Our qRT-PCR data demonstrated that FTO gene expression is relatively neuron-specific and notably is highly expressed in motor neurons (Fig. 3). By contrast, TBC1D1 would appear to be rather ubiquitously expressed.

Interestingly, long distance interactions have been reported for the FTO intronic region: (i) obesity-associated non-coding sequences within FTO interact with the promoter regions of IRX3 and FTO in human, mouse, and zebrafish, and (ii) obesity-associated FTO gene variants influence IRX3 expression in the human brain. The latter gene was associated with body mass, as IRX3-deficient mice displayed a decrease in their body weight by 25–30% [42]. Nutritional surveillance of ALS patients has been well established to be of fundamental importance, both in bulbar and spinal onset patients, and nutritional status is a prognostic factor for survival in ALS patients [16]. Jawaid et al. [22] reported that a decrease in body mass index is associated with faster progression of motor symptoms and shorter survival in ALS patients. Moreover, an LD block of 49 kb in the first intron of FTO contains numerous highly conserved non-coding elements (HCNEs) and lies within an extended region of conserved synteny involving the IRX3/5/6 cluster, the closest gene of which is the transcription factor-encoding IRX3 [37]. IRX3 is a member of the Iroquois homeobox gene family and plays a role in the early stages of neural development [7].

We also identified an example of C9orf72 hexanucleotide expansion in 1 of 28 patients with sALS (3.5%; Additional file 1: Table S4). This female patient, harboring this expansion, was diagnosed with unequivocal ALS but was lacked a positive family history of ALS. This massive hexanucleotide repeat expansion mutation in C9orf72, which has been linked to fALS, sALS, and frontotemporal dementia, has a single founder who is thought to have originated approximately 6300 years ago in Europe [31, 43]. Our findings differ somewhat from those reported by Mok and coworkers [35], the only study to date in fALS and sALS patients of Greek descent, in which 11 of 136 sALS patients (8.2%) were carriers of the expansion. Such a discrepancy may reflect inter-individual differences not as far as statistical power is concerned, but rather related to the patients’ genetic background, as the aforementioned study examined only individuals from Athens, whereas we recruited individuals from across the entire Greek mainland (north, central, and south Greece). Differential exposure to environmental factors are known to contribute to an increased risk of ALS; thus, exposure to lead, mercury, selenium, zinc, and copper is known to increase the risk of ALS [46], while high-risk populations include those who collect their domestic water from surface water [8], possibly reflecting an association between the neurotoxin b-methylamino-l-alanine (BMAA) produced by cyanobacteria and the risk of ALS. Al-Chalabi and Hardiman [4] have proposed a disease model, according to which ALS is the net result of environmental risks and time acting on a pre-existing genetic load, followed by an automatic, self-perpetuating decline until death. Akimoto et al. [2] reported marked differences in genotyping results for GGGGCC-repeat expansions in C9orf72. Herein, amplicon length analysis and repeat-primed PCR were combined as a recommended minimum requirement in a research setting [2].

Small sample sizes and data variability, plus our ancestrally diverse patient cohorts, should be taken into account when interpreting our findings. Yet, our datasets may assist future efforts of replicating these findings in independent ALS patient cohorts.

There is no single predominant genetic association underlying sALS. Instead, sALS may be considered to be the net result of the complex interplay of environmental factors and numerous low-risk susceptibility loci. We propose that under this polygenic model, a large number of alleles, each conferring a small genotypic risk, combine additively or multiplicatively to confer a range of susceptibilities in the general population [21, 47].

To our knowledge, this has been the first study to present a possible association between FTO variants and the genetic basis of sALS. These findings need to be further validated in a larger sALS patient cohort of Greek origin to overcome the small sample size bias, while a gene ontology analysis to look at links between FTO and the other genes known to be associated with ALS could support the validity of these findings.