A simplified protocol for gene expression-based biological dosimetry using peripheral whole blood.

PURPOSE: Assessing alterations in the expression of radiation-responsive genes in peripheral blood cells is considered a promising approach for high-throughput radiation biodosimetry. However, optimization of conditions for storage and transport of blood samples would be critical for obtaining reliable results. Recent studies involved the incubation of isolated peripheral blood mononuclear cells (in cell culture medium) and/or use of RNA stabilizing agents for sample storage, immediately after the ex vivo irradiation of whole blood. We used a simpler protocol by incubating undiluted peripheral whole blood without any RNA stabilizing agent, and studied the impact of storage temperature and incubation time on the expression levels of 19 known radiation responsive genes. MATERIALS & METHODS: Peripheral whole blood was γ-irradiated ex vivo at room temperature at low (0.5 Gy), moderate (1 Gy, 2 Gy) and high (4 Gy) doses and immediately incubated at two different temperatures at 4 °C or 37 °C for 2h, 4h and 24 h. Using qRT-PCR, mRNA expression levels of CDKN1A, DDB2, GADD45A, FDXR, BAX, BBC3, MYC, PCNA, XPC, ZMAT3, AEN, TRIAP1, CCNG1, RPS27L, CD70, EI24, C12orf5, TNFRSF10B, ASCC3 were analyzed at respective time-points and compared with the sham-irradiated controls. RESULTS: Transcriptional responses of all 19 genes did not alter significantly upon incubation of whole blood samples at 4 °C, as compared to untreated controls. However, incubation at 37 °C for 24 h resulted in significant radiation-induced overexpression in 14 out of the 19 genes analyzed (except CDKN1A, BBC3, MYC, CD 70 and EI24). Detailed patterns during incubation at 37 °C revealed time-dependent up-regulation of these genes, with DDB2 and FDXR showing significant up-regulation both at 4 and 24 h with the highest fold-change observed. CONCLUSION: Overall, the undiluted whole blood incubated at 37 °C for 24 h was found to elicit most optimal transcriptional response in the genes studied, with most profound overexpression of DDB2 and FDXR. We propose that sample storage/transport/post-transit incubation at the physiological temperature for up to 24 h may enhance the sensitivity of gene expression based biodosimetry and facilitate its usage for triage application.

Positive Association of D Allele of ACE Gene With High Altitude Pulmonary Edema in Indian Population.

OBJECTIVE: High altitude pulmonary edema (HAPE) is a potentially fatal high altitude illness occurring as a result of hypobaric hypoxia with an unknown underlying genetic mechanism. Recent studies have shown a possible association between HAPE and polymorphisms in genes of the renin-angiotensin-aldosterone system (RAAS), which play a key role in sensitivity of an individual toward HAPE. METHODS: For the present investigation, study groups consisted of HAPE patients (HAPE) and acclimatized control subjects (rCON). Four single-nucleotide polymorphisms (SNPs) were genotyped using restriction fragment length polymorphism (RFLP) analysis in genes of the RAAS pathway, specifically, renin (REN) C(-4063)T (rs41317140) and RENi8-83 (rs2368564), angiotensin (AGT) M(235)T (rs699), and angiotensin-converting enzyme (ACE) insertion/deletion (I/D) (rs1799752). RESULTS: Only the I/D polymorphism of the ACE gene showed a significant difference between the HAPE and rCON groups. The frequency of the D allele was found to be significantly higher in the HAPE group. Arterial oxygen saturation levels were significantly lower in the HAPE group compared with the rCON group and also decreased in the I/D and D/D genotypes compared with the I/I genotype in these groups. The other polymorphisms occurring in the REN and AGT genes were not significantly different between the 2 groups. CONCLUSIONS: These findings demonstrate a possible association of the I/D polymorphism of the ACE gene with the development of HAPE, with D/D being the at-risk genotype.

High-altitude pulmonary edema: review.

OBJECTIVE: At High altitude (HA) (elevation >2,500 m), hypobaric hypoxia may lead to the development of symptoms associated with low oxygen pressure in many sojourners. High-altitude pulmonary edema (HAPE) is a potentially fatal condition, occurring at altitudes greater than 3,000 m and affecting rapidly ascending, non-acclimatized healthy individuals. It is a multifactorial disease involving both environmental and genetic risk factors. Since thousands of lowlanders travel to high altitude areas for various reasons every year, we thought it would be interesting to review pathological aspects related to hypobaric hypoxia, particularly HAPE. METHOD: Since the pathogenesis of HAPE is still a subject of study, we systematically identified and categorized a broad range of facets of HAPE such as its incidence, symptoms, physiological effects, pathophysiology including physiological and genetic factors, prevention and treatment. RESULTS: This review focuses on HA-related health problems in general with special reference to HAPE, which is one of the primary causes of deaths at extreme altitudes. Hence, it is extremely important, as it summarizes the literature in this area and provides an overview of this severe HA malady for evaluation of physiological, biochemical and genetic responses during early induction and acclimatization to HA. This article could be of broad scientific interest for researchers working in the field of high altitude medicine.

Distribution of performance-related gene polymorphisms (ACTN3 R577X and ACE ID) in different ethnic groups of the Indian Army.

BACKGROUND: India has a heterogeneous geographical location with varying ethnic groups with distinct genetic pools. Polymorphisms R577X and insertion/deletion (I/D), occurring in α-actinin3 (ACTN3) and angiotensin converting enzyme (ACE) genes, respectively, are reportedly associated with sprint and endurance performance. The normative genetic background of a population provides the baseline genetic diversity of the population. METHODS: We investigated the distribution of R577X and I/D polymorphisms in four Indian ethnic populations employed in the Indian Army that requires high levels of physical fitness. A total of 598 army subjects with four different ethnicities were recruited for the study: Rajputs, South Indians, Gorkhas, and Ladakhis. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism followed by statistical analysis. RESULTS: The present study reports for the first time the genotypic distribution of ACTN3 R577X polymorphism in four ethnic groups of the Indian population with no significant change in distribution of R and X alleles among these groups and their overall percentage bearing similarity with the Caucasian population. ACE I/D polymorphism showed significant differences between Rajputs and Gorkhas, Rajputs and Ladakhis, Gorkhas and South Indians, and South Indians and Ladakhis. Combined genotypic analysis showed the highest frequencies of ID+RX in all ethnic groups. CONCLUSIONS: Our study reports the genetic predominance of ACE I allele in Gorkhas and I allele predominance in Ladakhis for the first time, thus suggesting that they could be better endurance performers. Both these ethnic groups evolved from the Mongolian race and hence could have similar genotypic distribution. This study provides a comparative account of the Indian normative genetic data in performance-related genes.

Association of polymorphisms in angiotensin and aldosterone synthase genes of the renin-angiotensin-aldosterone system with high-altitude pulmonary edema.

Studies on different populations have suggested variability in individual susceptibility to altitude sickness depending on genetic makeup. The renin-angiotensin-aldosterone system (RAAS) pathway plays a key role in regulation of vascular tone and circulatory homeostasis. The present study was undertaken to investigate the possible association of the RAAS in the development of high-altitude pulmonary edema (HAPE) in lowlanders exposed to high altitude. Three categories of subjects were selected: individuals who developed HAPE on acute induction to high altitude (HAPE); individuals tolerant to high-altitude exposure who showed no symptoms of HAPE (resistant controls; rCON); and natives of high altitude (HAN). Genetic variants in the genes of the RAAS such as renin (REN), angiotensin (AGT), angiotensin-converting enzyme (ACE), aldosterone synthase (CYP11B2) and angiotensin II receptor type 1 (AGTR1) have been investigated. The T174M polymorphism in AGT showed a significant difference in HAPE and HAN and also HAN and controls. Also, genotyping in the CYP11B2 T-344C promoter region resulted in a significant difference between HAPE and HAN both at genotypic and allelic levels. The genotypic difference was statistically insignificant for the AGTR1 A1166C 3' UTR. The present investigation demonstrates a possible association between the polymorphisms existing in the RAAS pathway T174M and CYP11B2 C-344T and sensitivity of an individual to develop HAPE. The results also indicate the existence of ethnic variation between the HAN and the other two groups comprising lowlanders.