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The Glu298Asp polymorphism in the NOS3 gene is not associated with sporadic Alzheimer's disease
  4. I MATEO,
  1. Neurology Service, Marqués de Valdecilla, University Hospital, Santander, Spain
  2. Division of Preventive Medicine, University of Cantabria School of Medicine, Santander, Spain
  1. Dr O Combarros combarro{at}
  1. Neurology Service, Marqués de Valdecilla, University Hospital, Santander, Spain
  2. Division of Preventive Medicine, University of Cantabria School of Medicine, Santander, Spain
  1. Dr O Combarros combarro{at}

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Nitric oxide (NO) production by microglial cells, astrocytes, and brain microvessels is enhanced in patients with Alzheimer's disease, and there is a growing evidence that NO is involved in neuronal death in Alzheimer's disease.1 The oxidative stress caused by NO in the brain could be genetically regulated, and NO synthase (NOS) genes could modulate the susceptibility of developing the disease. Two isoforms of NOS, inducible (NOS2) and endothelial (NOS3), have been examined in Alzheimer's disease. A pentanucleotide repeat polymorphism within the promoter region of the NOS2 gene is not associated with Alzheimer's disease but may be a predisposing factor in the development of dementia with Lewy bodies.2 Similarly, a rare polymorphism in a 27 base pair repeat in intron 4 of NOS3 is not linked to an increased risk of developing Alzheimer's disease.3 On the other hand, Dahiyat et al 4 analyzed a missense Glu298Asp variant in exon 7 of the NOS3 gene in a British population sample and found an increased frequency of the Glu/Glu genotype among patients with Alzheimer's disease compared with controls, and interaction with the APOE gene was also found. However, a recent American series5 disclosed no significant increase in the risk of Alzheimer's disease with homozygosity for the Glu allele in the Glu298Asp polymorphism. Thus, we investigated the association of this polymorphism and Alzheimer's disease in a Spanish case-control study.

The study included 301 patients (66% women; mean age at censoring 75.2 years (SD 9.0), range 50 to 98 years; mean age at onset 71.5 years (SD 8.9), range 40 to 95 years) who met National Institute of Neurological and Communicative Disorders Association criteria for probable Alzheimer's disease.6 Control subjects were 309 unrelated patients (71% women; mean age 80.3 years (SD 7.7), range 63 to 99 years) randomly selected from a nursing home; these subjects underwent complete neurological and medical examinations, which showed that they had no significant illness and had mini mental state examination scores of 28 or greater, verified by at least one subsequent annual follow up assessment. The patients with Alzheimer's disease and control subjects were white, originating from a limited geographical area in northern Spain. Genotyping of Glu298Asp NOS3 and APOE were performed by polymerase chain reaction followed by restriction endonuclease digestions.4 Qualitative factors were analyzed estimating the odds ratio (OR) and its 95% confidence interval (95% CI). Means of quantitative variables were compared using the Student'st test. All statistical analyses were performed with the statistical package “Stata Intercooled version 6” (Stata Corporation, College Station, TX, USA).

The distribution of Glu298Asp NOS3 genotypes and alleles in patients with Alzheimer's disease followed the Hardy-Weinberg equilibrium and did not differ significantly from that of the controls (table 1). The OR for the Glu/Glu genotype compared with Glu/Asp and Asp/Asp genotypes was 0.98 (95% CI 0.70–1.37) (p=0.90). Moreover, when Alzheimer's disease was categorised by age of onset in early onset (<70 years) and late onset (⩾70 years) subsets, no increased risk of the disease was found in homozygotes for the Glu allele in each age category. The same results held when our data set was stratified by sex. Next, we split the data based on APOE carrier status. There was no significant association between the Glu/Glu genotype and the risk of Alzheimer's disease in either APOE ε4 carriers (OR 0.87 (95% CI 0.47–1.61), p=0.66) and non-carriers (OR 0.92 (95% CI 0.58–1.45), p=0.72). Our analysis showed the expected association between the APOE ε4 allele and Alzheimer's disease (OR 5.28 (95% CI 3.67–7.59)) for carrying one or two copies of ε4; however, this risk was not increased by the presence of the Glu/Glu genotype (OR with APOE ε4 plus Glu/Glu 4.71 (95% CI 2.72–8.16)).

Table 1

Glu298Asp NOS3 genotype and allele frequencies in patients with Alzheimer's disease (AD) and control subjects

The present study failed to replicate the findings of Dahiyatet al,4 and there were no significant differences in the percentage of patients with Alzheimer's disease and controls with the Glu/Glu genotype, even after stratifying by age, sex, and APOE carrier status. The failure to detect an association between Alzheimer's disease and homozygosity for the Glu allele in codon 298 of NOS3 cannot be ascribed to lack of statistical power because our sample of 301 patients and 309 controls had the power to detect an OR of at least 1.7 assuming a significance level (α) of 0.05, a power (1-β) of 0.80, and an exposure frequency of 0.33 in controls (those having the Glu/Glu genotype). By comparison, the OR for the Glu/Glu genotype in the British sample4 was 2.04. The most likely interpretation of this discrepancy would be an ethnic difference in Alzheimer's disease susceptibility associated with the polymorphism. In fact, it is noteworthy that the Glu allele frequency of our Spanish controls (56%) was less than those reported for the controls from the United kingdom4 (65%) and the United States5 (71%). On the other hand, the frequency of the Glu/Glu genotype in our patients with Alzheimer's disease, which was similar to the frequency in our controls, was less than the corresponding frequencies in British4 and North American5 patients by 25% and 13%, respectively. These findings suggest that the frequency of the Glu298Asp polymorphism may vary considerably within different ethnic groups.

Although our data confirm the well established association between APOE ε4 and Alzheimer's disease, our results do not support the notion that the Glu/Glu genotype in the 298 codon of NOS3 gene is a risk factor for Alzheimer's disease, neither through an independent effect nor through interaction with the existing APOE ε4 risk.