High altitude pulmonary oedema (HAPE) is a severe form of altitude illness that develops in travellers on rapid ascent to or physical exertion at altitudes of >2500 m.1 The disease is characterised by pulmonary hypertension, uneven vasoconstriction, and overperfusion which is thought to cause stress failure of pulmonary capillaries leading to alveolar flooding.1 Since uneven pulmonary vasoconstriction appears to play an important part in the development of HAPE, the genes involved in maintaining pulmonary vascular tone—for example, angiotensin converting enzyme (ACE) and endothelin‐1 (ET‐1)—could be possible candidates for HAPE.
Earlier studies showed that the selective pressure of hypobaric hypoxia acted in favour of those alleles of ACE and ET‐1 which were beneficial in maintaining a healthy state at high altitude.2,3 On the other hand, unfavourable alleles are likely to contribute to the susceptibility to HAPE. This hypothesis lends support to an earlier report on the allelic variants of endothelial nitric oxide synthase gene.4
We therefore investigated ACE insertion/deletion (I/D) (GenBank accession no X62855) and ET‐1 5′‐untranslated region (UTR) microsatellite (CT)n‐(CA)n (GenBank accession no J05008), −3A/−4A (rs10478694), G2288T (rs2070699), and Lys198Asn (rs5370) polymorphisms in 64 patients with HAPE (HAPE‐p) and 53 HAPE resistant controls (HAPE‐r). The HAPE‐r were healthy individuals who had climbed 2–3 times to altitudes greater than 3500 m and carried out routine strenuous physical activities without suffering from HAPE; in contrast, the other group suffered from HAPE on their very first visit. The study groups consisted of age matched (30–40 years) individuals of the same ethnicity. An institutional review committee approved the investigation and all subjects gave informed consent.
HAPE was diagnosed on the basis of the criteria described earlier.4 After recovery the HAPE‐p were examined to exclude the possibility of any previous cardiopulmonary diseases. The subjects were genotyped for the five polymorphisms of the two genes using primers and conditions shown in table S1 (available online at http://www.thoraxjnl.com/supplemental). The plasma ACE levels were measured by a kinetic method using N‐(3‐[2‐furyl] acryloyl)‐Phe‐Gly‐Gly as substrate. The plasma ET‐1 levels were determined by ELISA (Assay Designs, Ann Arbor, USA). SPSS statistical software for Windows (release 10), EPIINFO 6, and SNP Alyze program (Version 3.1, Dynacom, Mobara‐shi, Japan) were used to perform the statistical analysis.
The mean (SD) ACE activity and ET‐1 levels were significantly higher in HAPE‐p than in HAPE‐r (84.6 (26.2) v 40.7 (12.1) U/l and 8.0 (2.5) v 3.5 (0.7) pg/ml, respectively; both p<0.0001). Furthermore, a direct relationship was observed between ACE activity and ET‐1 levels in HAPE‐p and HAPE‐r (r = 0.31, p = 0.03 and r = 0.32, p = 0.02, respectively), which reflects their interaction. ACE generates angiotensin II which induces ET‐1 transcription and secretion in vitro in a variety of cell types including endothelial and vascular smooth muscle cells.5 ET‐1 is also involved in the regulation of ACE activity in vivo independently of ACE expression.6
The polymorphisms were in Hardy‐Weinberg equilibrium in both groups and are shown in table 1. The ID+DD and GT+TT genotypes of ACE I/D and ET‐1 G2288T polymorphisms were over‐represented in HAPE‐p (p = 0.03 and p = 0.002, respectively), with D and T alleles being more frequent in HAPE‐p than HAPE‐r. The (CT)n‐(CA)n repeats were segregated and recognised as shorter (13–30) and longer (31–45) based on our earlier observation.3 However, unlike in our previous report, the shorter and longer repeats did not correlate with ET‐1 levels. Analysis of the possible genotype combinations between the five polymorphisms showed that there were significantly fewer genotype combinations II/longer repeats and II/GG between the two genes in HAPE‐p than in HAPE‐r (p = 0.02 and p = 0.002, respectively). The longer repeats/Lys198Lys genotype combination within ET‐1 was significantly less in HAPE‐p than in HAPE‐r (1% v 9%, odds ratio 0.10 (95% CI 0.01 to 0.82), p = 0.01). Our findings support the hypothesis that ACE and ET‐1 polymorphisms have a role in the susceptibility to HAPE. The findings of the present study and earlier reports on adaptation to high altitude2,3 together indicate that ACE and ET‐1 genes are significant in high altitude physiology.
Table 1 Distribution of ACE I/D and ET‐1 −3A/−4A, G2288T and Lys198Asn polymorphisms and their combinations in the HAPE‐p and HAPE‐r.
| Genotypes/genotype combinations* | HAPE‐p (n = 64) | HAPE‐r (n = 53) | χ2 | p value | OR (95% CI) |
|---|---|---|---|---|---|
| ACE | |||||
| II | 18 (28) | 23 (43) | |||
| ID | 34 (53) | 21 (40) | |||
| DD | 12 (19) | 9 (17) | 5.10 | 0.07 | |
| ID+DD | 46 (72) | 30 (57) | 4.91 | 0.03 | 1.94 (1.08 to 3.50) |
| ET‐1 | |||||
| Longer repeats† | 17 (27) | 19 (35) | |||
| Shorter repeats† | 47 (73) | 34 (65) | 1.15 | 0.28 | 1.46 (0.80 to 2.66) |
| −3A/−3A | 44 (69) | 35 (66) | |||
| −3A/−4A | 20 (31) | 18 (34) | 0.21 | 0.65 | 0.87 (0.48 to 1.58) |
| GG | 15 (23) | 23 (43) | |||
| GT | 37 (58) | 22 (42) | |||
| TT | 12 (19) | 8 (15) | 9.09 | 0.01 | |
| GT+TT | 49 (77) | 30 (57) | 9.05 | 0.002 | 2.53 (1.37 to 4.65) |
| Lys198Lys | 22 (34) | 17 (31) | |||
| Lys198Asn | 32 (50) | 27 (52) | |||
| Asn198Asn | |||||
| Lys198Asn+ | 10 (16) | 9 (17) | 0.20 | 0.90 | |
| Asn198Asn | 42 (66) | 36 (69) | 0.65 | 0. 76 | 0.87 (0.48 to 1.58) |
| ACE+ET‐1 | |||||
| II/Longer repeats | 1 (1) | 4 (8) | 0.12 (0.01 to 0.95) | ||
| Remaining combinations | 63 (99) | 49 (92) | 5.70 | 0.02 | |
| II/−3A/−3A | 13 (21) | 15 (28) | 0.68 (0.36 to 1.31) | ||
| Remaining combinations | 51 (79) | 38 (72) | 1.32 | 0.25 | |
| II/GG | 5 (7) | 12 (23) | 0.25 (0.10 to 0.62) | ||
| Remaining combinations | 59 (93) | 41 (77) | 10.04 | 0.002 | |
| II/Lys198Lys | 9 (14) | 8 (16) | 0.85 (0.39 to 1.86) | ||
| Remaining combinations | 55 (86) | 45 (84) | 0.15 | 0.69 |
HAPE‐p, individuals with high altitude pulmonary oedema; HAPE‐r, individuals resistant to high altitude pulmonary oedema.
The genotypes and genotype combinations are presented as number (%).
*Genotype combinations were grouped into wild‐type genotype combinations and remaining combinations.
†(CT)n‐(CA)n repeats were segregated and recognised as shorter (13–30) and longer (31–45).
In conclusion, this study showed that ACE and ET‐1 variants have independent and interactive roles in the susceptibility to HAPE. Higher ACE activity and ET‐1 levels correlated with HAPE. The ACE I/D and ET‐1 G2288T polymorphisms emerged as noteworthy variants showing association with HAPE. Owing to the small sample size, the difference in representation of the polymorphisms and their correlation with biochemical parameters did not reach greater statistical significance; in particular, we consider our data on the genotype combinations between the polymorphisms of the two genes to be preliminary. Nevertheless, since HAPE samples are difficult to obtain, the findings of this study are important and warrant confirmation in a larger sample. The results of this study may find application in identifying individuals with a predisposition to HAPE.
Details of the primers and conditions for genotyping the five polymorphisms are shown in table S1 available on the Thorax website at http://www.thoraxjnl.com/supplemental.
Copyright © 2006 BMJ Publishing Group and British Thoracic Society
Acknowledgements
The authors thank Ven Thubten Choegyal, Chairman, Ladakh Heart Foundation who helped with the collection of blood samples and Professor S K Brhamachari for his support and encouragement.
Footnotes
This work was funded by the Council of Scientific and Industrial Research.
Competing interests: none.
Details of the primers and conditions for genotyping the five polymorphisms are shown in table S1 available on the Thorax website at http://www.thoraxjnl.com/supplemental.
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