Screening of the transcriptional regulatory regions of vascular endothelial growth factor receptor 2 (VEGFR2) in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, in which loss of motor neurones in the spinal cord, brainstem and cerebral cortex causes progressive paralysis. Ten percent of cases are familial, and one fifth of these are caused by a mutation in the gene encoding superoxide dismutase (SOD1). In the majority of familial and sporadic cases of ALS, the cause of selective loss of motor neurones is unknown, but there is growing evidence of the importance of dysregulation of vascular endothelial growth factor (VEGF) as a factor in motor neurone degeneration.

VEGF is an endothelial cell mitogen essential for angiogenesis, and upregulated by hypoxia. In 2001, Oosthuyse et al found that deletion of the hypoxia response element of the VEGF gene in mice caused motor neurone degeneration with clinical and pathological features similar to ALS [1]. VEGF was subsequently shown to act as a neurotrophic factor in vitro and in vivo [1, 2]. VEGFR2 (also known as KDR) is the major agonist receptor of VEGF, and has been shown to mediate its neuroprotective effects in vitro [1]. Neuronal overexpression of VEGFR2 in SOD1 transgenic mice delays the onset of motor impairment, and prolongs survival [3]. A recent immunohistochemical study showed that the expression of both VEGF and VEGFR2 was downregulated on anterior horn cells, and VEGFR2 immunostaining in the neuropil was decreased in the spinal cord in patients with ALS, compared to normal controls [4].

The role of VEGFR2 in the mediation of the neurotrophic effects of VEGF, and its reduced expression in neural tissue of ALS patients identify it as an important candidate gene in ALS. We have sequenced the 5' UTR and promoter region of the VEGFR2 gene in patients with ALS, to determine whether the observed downregulation of VEGFR2 expression in ALS patients is related to the segregation of certain alleles at polymorphic sites within the regulatory regions of the VEGFR2 in the ALS population. We have screened the regulatory regions and 4 exons of functional significance in the VEGFR2 gene for mutations in ALS patients, which are not present in control populations. These 4 exons were identified for screening both on the basis of their functional significance [5], and the presence of previously published polymorphisms within the coding regions [6]

All patients and controls were from Caucasian backgrounds. 58% of patients were male, and the mean age was 61.8. 8.6% of patients had one of the clinical variants of ALS (5.6% PMA, 2% PLS and 1% PBP). In the control population, 56% were male, and the mean age was 56.6.

Exons 7, 18, 21 and 27 of the VEGFR2 gene were screened for mutations or polymorphisms in 100 ALS patients. Exon 7 lies within the VEGF binding domain and exons 18 and 21 within the protein kinase domain. The function of exon 27 is unknown, but it is a region that is highly conserved across species. No mutations were identified in the exons or intron/exon boundaries of exons 7, 18, 21 or 27. The previously described polymorphisms in exons 18, 21 and 27 were not present in this population of ALS patients. The A allele at position +1192 in exon 7 was present at a frequency of 0.06 in both ALS and control populations (Table 2), consistent with its published frequency [6].

The VEGFR2 5' UTR and promoter region was sequenced from -315 to +302 relative to the transcription start site, which includes the core promoter region of the VEGFR2 gene (Figure 1) [7], in 301 ALS patients and 239 unrelated neurologically normal controls. Five polymorphisms were identified in these regulatory regions, of which three were novel (305 C/T, -263 C/T, and -57 C/T). Previously described polymorphisms [6] -123C/G and -17A/T were not seen. There was no difference in frequency of polymorphisms -305 C/T, -263 C/T, -65 C/T and +31 A/G in the ALS population, relative to the control population (Table 2). The novel polymorphism -57 C/T is located in a consensus sequence for binding SP1, the major transcriptional regulator of VEGFR2 [8, 9], and was found in one patient and one control case (Figure 2)

The VEGF signalling pathway has an important role in the maintenance of neuronal survival, and dysregulation of this pathway leads to motor neurone death. A meta-analysis of pooled European populations demonstrated that two haplotypes of functional polymorphisms in the 5' UTR of the VEGF gene conferred a 1.8 fold greater risk of ALS [2], although in other populations, no association between VEGF polymorphisms and ALS is seen [2, 10, 11]. This does not exclude the possibility that dysregulation of the VEGF signalling pathway plays a role in the pathogenesis of ALS in these patients, as genetic factors may also control response to VEGF [12]. We have previously shown that expression of VEGFR2 is downregulated in the CNS of ALS patients [4]. In this study we have addressed the question of whether sequence variation in the regulatory regions of the VEGFR2 gene in ALS patients may contribute to lower expression of this VEGF agonist receptor. We have sequenced important functional regions of this candidate gene, to determine whether sequence variation in the coding regions could affect function of the receptor, and therefore responsiveness to VEGF in ALS.

No mutations or differences in polymorphism frequency were identified in the ALS population, in the regulatory regions or 4 coding regions of probable functional significance. These findings do not support the hypothesis that genetic factors reduce VEGF responsiveness in ALS, via downregulation of expression or function of VEGFR2. This study had a power of 89% to detect a relative risk of 1.8 at 0.05 significance level, therefore the lack of association between VEGFR2 promoter region polymorphisms and ALS is unlikely to be due to sample size.

Mechanisms other than genetic factors may reduce VEGFR2 expression in ALS. In ALS patients without 'at-risk' VEGF haplotypes, decreased plasma levels of VEGF were also identified [2]. Similarly, reduced expression of VEGFR2 may occur secondary to other disease mechanisms, such as gene downregulation due to dysregulation of transcription factors, which is seen in a mutant SOD1 cell model of ALS [13]. VEGFR2 is a large gene with 30 exons, and although we selected functionally significant regions of the gene, we have not excluded the possibility that sequence variation in other exonic regions could disrupt the function of this protein in ALS patients.

There is no evidence from sequencing of the regulatory regions and 4 functionally significant exonic regions of the VEGFR2 that genetic variation in this agonist receptor causes downregulation of VEGF signalling in ALS patients.