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High altitude pulmonary oedema (HAPE) is a severe form of altitude illness that may develop in individuals on rapid ascent to altitudes above 2500 m.1 The disease is characterised by hypoxia induced pulmonary vasoconstriction caused by endothelial dysfunction and intravascular fluid retention.2,3 While some families and individuals are at risk, those with a long ancestry at high altitude have a lower risk. Moreover, individuals who have had HAPE are at a greater risk of repeat events. Such data support a strong genetic component to HAPE susceptibility, perhaps associated with a founder effect. It is likely that long term exposure to high altitude provides a natural positive adaptive pressure to alleles that prevent the illness. We hypothesise that allelic variants at the same locus in a gene are involved in adaptation and HAPE.
We therefore investigated the Glu298Asp and 4b/4a polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and −344T/C, intron-2 conversion and Lys173Arg polymorphisms of the aldosterone synthase gene (CYP11B2) in 59 patients with HAPE who developed the disease at 3400 m, 64 lowland controls (LLs) who had been to the same altitude two or three times and even to 5600 m, and 136 highland natives (HLs) from Leh, Ladakh (3400 m). The study groups consisted of unrelated and age matched men aged 30–40 years who had been inhabitants of their respective lands since ancient times. The HAPE patients and LLs were of the same ethnic origin and ascended in a similar manner. The diagnosis of HAPE was based on chest radiographs and other clinical symptoms. Blood samples were collected in the morning in the supine position after overnight fasting. Subjects abstained from smoking for 12 hours before sample collection. The institutional ethical committee approved the investigation and all subjects gave informed consent.
Genotype determination of the five polymorphisms in the two genes was performed by modified cycling conditions. Genotypes were randomly validated on a 377 DNA sequencer (Applied Biosystems, USA). Plasma nitric oxide (NO) estimated as nitrite by the enzymatic Griess method (Calbiochem, USA) and aldosterone levels were determined by radioimmunoassay (Immunotech, France). SPSS software for windows (release 10.0) was used for the statistical analysis.
This study is the first to report plasma NO and aldosterone levels in patients with HAPE and HLs. NO levels were significantly lower in the HAPE group (46.17 (13.94) µM) than in HLs (95.35 (27.56) µM) or LLs (90.53 (29.97) µM) (p<0.0001 for each). The NO levels in the order HLs > LLs > HAPE support earlier reports of impaired NO synthesis in HAPE4 and increased NO levels in mountain dwellers.5 Previous studies, however, measured the exhaled NO level which is not the exact measure of endogenous NO production. The highest NO levels in HLs signify its importance in the maintenance of regular physical activity at high altitude. NO improves the ventilation/perfusion ratio and lowers the alveolar to arterial oxygen tension difference by increasing oxygen saturation. The levels of aldosterone in the HAPE group (467.0 (339.0) pmol/l) were significantly higher in the HLs (376.3 (169.5) pmol/l; p = 0.05), LLs (155.5 (109.9) pmol/l; p<0.0001), or both (p<0.0001). This finding is in agreement with the hypothesis that antidiuresis followed by fluid retention is one of the mechanisms leading to HAPE,3 in which aldosterone plays a pivotal role. NO inhalation therapy and the use of diuretics to treat HAPE2 support the decreased levels of endogenous NO and increased levels of aldosterone observed in the present study.
The three groups were in Hardy-Weinberg equilibrium for the polymorphisms. The genotype and allele frequency analysis of the Glu298Asp and 4b/4a polymorphisms of the eNOS gene revealed that the Asp and 4a alleles were over-represented in the HAPE group and that the Glu and 4b alleles were over-represented in the HLs (table 1, above). A recent study also reported an association of mutant alleles with the disorder.6 The presence of the Asp variant renders the enzyme susceptible to intracellular proteases.7 Proteolysis may reduce NO levels which may lead to impaired vasodilation and endothelial dysfunction in a hypoxic environment, increasing susceptibility to HAPE. The over-representation of wild-type alleles in HLs suggests that the mutant alleles associated with HAPE are eliminated in HLs as a process of natural selection. Indeed, the tolerance of Himalayan populations to hypoxia, which is reflected in their metabolic and physiological traits, is believed to be the result of adaptation.8 In the case of CYP11B2 polymorphisms, the intron-2 conversion homozygotes were over-represented in the HAPE subjects compared with HLs (p = 0.03) whereas the −344T/C and Lys173Arg polymorphisms were not associated with the disorder (data not shown).
Our results suggest a significant role for NO and aldosterone in the pathogenesis of HAPE. The over-representation of eNOS Asp and 4a alleles in patients with HAPE associates these alleles with the disorder, whereas over-representation of Glu and 4b alleles in HLs suggests that they have a role in adaptation to high altitudes. These findings suggest, for the first time, that allelic variants at the same locus are involved in HAPE and adaptation.
AA and RC contributed equally.
Supported by grants from the Council of Scientific and Industrial Research.
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